Expression dynamics of the genes for the hypothalamo-pituitary-gonadal axis in tiger puffer (Takifugu rubripes) at different reproductive stages.

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.

Light-induced and circadian expressions of melanopsin genes opn4xa and opn4xb in the eyes of juvenile grass puffer Takifugu alboplumbeus.

Animals regulate a variety of aspects of physiology according to environmental light conditions via nonvisual opsins such as melanopsin. In order to study photic regulation of fish physiology, expression changes of the genes for melanopsin (opn4xa and opn4xb) and effects of light on them were examined in juvenile grass puffer Takifugu alboplumbeus using quantitative real-time PCR. In the brain of juvenile fish, no significant diurnal nor circadian changes were observed in opn4x mRNA levels. On the other hand, in the eyes, the mRNA level of opn4xa showed a significant diurnal rhythm with a peak at Zeitgeber time (ZT) 4, while no apparent circadian changes were observed. The mRNA level of opn4xb in the eyes showed a diurnal change similar to that of opn4xa, while it showed a significant circadian change. Furthermore, continuous exposure to light during a subjective night significantly increased the mRNA levels of opn4xa in the eyes at ZT24, suggesting that light induces gene expression of opn4xa in the eyes and that the induction occurs only during the night-day transition period. These results suggest that Opn4xa and Opn4xb play differential roles in the eyes of juvenile grass puffer to mediate the physiological effects of environmental light information.

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish.

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10-7M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10-9 to 10-7M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10-7M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

Tetrodotoxin Detection in Japanese Bivalves: Toxification Status of Scallops.

Tetrodotoxin (TTX), or pufferfish toxin, has been frequently detected in edible bivalves around the world during the last decade and is problematic in food hygiene and safety. It was reported recently that highly concentrated TTX was detected in the midgut gland of the akazara scallop Chlamys (Azumapecten) farreri subsp. akazara collected in coastal areas of the northern Japanese archipelago. The toxification of the bivalve was likely to involve the larvae of the flatworm, Planocera multitentaculata. However, the overall status of bivalve TTX toxification has not been elucidated. In this study, 14 species/subspecies of bivalves from various Japanese waters were subjected to LC-MS/MS analysis to reveal TTX toxification state, demonstrating that the Pectinidae, including C. farreri akazara, Chlamys farreri nipponensis, Chlamys (Mimachlamys) nobilis, and Mizuhopecten yessoensis, accumulated TTX in their midgut gland. Many individuals of C. farreri akazara and C. farreri nipponensis were found with high concentrations of TTX, while C. nobilis and M. yessoensis exhibited low concentrations. The extent of TTX accumulation in C. farreri akazara and C. farreri nipponensis varied widely by region and season. Curiously, no other bivalve species investigated in this study showed evidence of TTX. These results suggest that monitoring for TTX, like other shellfish toxins, is necessary to ensure that pectinid bivalves are a safe food resource.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Adaptation to the shallow sea floor environment of a species of marine worms, Oligobrachia mashikoi, generally inhabiting deep-sea water.

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Seasonal variations in marine polycyclic aromatic hydrocarbons off Oki Island, Sea of Japan, during 2015-2019.

Concentrations of 13 phase-partitioned polycyclic aromatic hydrocarbons (PAHs) in seawater were monitored monthly off Oki Island, Japan, during 2015-2019 to elucidate seasonal variations, main source, and transport pathways of PAHs in the southwestern Sea of Japan. Total PAH (dissolved plus particulate) concentrations in surface seawater at 36°09.0'N, 133°17.3'E (site OK) were in the range 0.49-9.36 ng L-1 (mean 2.77, SD 2.05 ng L-1) with higher levels in summer-autumn, an order of magnitude lower than those in the East China Sea during 2005 and 2009-2011 and about one-third of those recorded in the Sea of Japan in 2008 and 2010. The main sources of dissolved and particulate PAHs were combustion products. Increasing dissolved PAH levels during July-October indicate that the area around southern Oki Island is impacted by PAH-rich summer continental-shelf water transported by the Tsushima Warm Current flowing from the East China Sea.

Temporal Variations of Polycyclic Aromatic Hydrocarbons in the Seawater at Tsukumo Bay, Noto Peninsula, Japan, during 2014-2018.

Concentrations of phase-partitioning 13 polycyclic aromatic hydrocarbons (PAHs) in seawater were investigated in the Tsukumo Bay, Noto Peninsula, Japan, during 2014-2018, to improve the understanding of the environmental behavior of PAHs in the coastal areas of the Japan Sea. Total PAH (particulate plus dissolved) concentrations in surface seawater were in the range 0.24-2.20 ng L-1 (mean 0.89 ng L-1), an order of magnitude lower than the mean values observed in the Japan Sea in 2008 and 2010. Although the PAH contamination levels during 2014-2018 were significantly lower than those in the East China Sea, the levels increased from 2014 to 2017 and were maintained at the higher level during 2017-2018. The main sources of particulate and dissolved PAHs during 2014-2018 were combustion products, of which the former were more influenced by liquid fossil-fuel combustion and the latter by biomass or coal combustion. The increase in particulate PAH concentrations in October-December during 2014-2018 was due to the impact of PAH-rich airmasses transported from the East Asian landmass in the northwesterly winter monsoon winds. The increase in dissolved PAH levels during July-September in 2014, 2016, 2017, and 2018 indicates that the Tsukumo Bay is possibly impacted by the PAH-rich summer continental shelf water transported by the Coastal Branch of the Tsushima Warm Current, which flows into the Japan Sea from the East China Sea.

Interannual Survey on Polycyclic Aromatic Hydrocarbons (PAHs) in Seawater of North Nanao Bay, Ishikawa, Japan, from 2015 to 2018: Sources, Pathways and Ecological Risk Assessment.

To improve the understanding of the emission sources and pathways of polycyclic aromatic hydrocarbons (PAHs) in the coastal environments of remote areas, their particulate and dissolved concentrations were analyzed on a monthly basis from 2015 to 2018 in surface waters of Nanao Bay, Japan. The concentration of the targeted 13 species of PAHs on the United States Environmental Protection Agency (USEPA) priority pollutant list in dissolved and particle phases were separately analyzed by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. Particulate and dissolved PAHs had average concentrations of 0.72 ng∙L-1 and 0.95 ng∙L-1, respectively. While most of the samples were lower than 1 ng∙L-1, abnormally high levels up to 10 ng∙L-1 were observed in the winter of 2017-2018 for particulate PAHs. Based on the isomer ratios of Flu to Flu plus Pyr, it was possible to determine that the pyrogenic loads were greater than the petrogenic loads in all but four out of 86 samples. The predominant environmental pathway for PAHs in winter was determined to be long-range atmospheric transportation fed by the East Asian winter monsoon, while for the summer, local sources were more relevant. By the risk quotients method, it was determined that PAHs in surface seawater presented a very low risk to marine life during the interannual survey.