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Nagata T.,Kumamoto Prefectural Fisheries Research Center | Nagata T.,Kumamoto University | Sameshima M.,Kumamoto Prefectural Fisheries Research Center | Uchikawa T.,Kumamoto University | And 2 more authors.
Fisheries Science | Year: 2017

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters. © 2017 Japanese Society of Fisheries Science


Nishitani G.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Nagai S.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Baba K.,Hokkaido Hakodate Fisheries Experiment Station | Kiyokawa S.,Hokkaido Abashiri Fisheries Experiment Station | And 6 more authors.
Applied and Environmental Microbiology | Year: 2010

We analyzed cryptophyte nucleomorph 18S rRNA gene sequences retained in natural Myrionecta rubra cells and plastid 16S rRNA gene and psbA sequences retained in natural cells of several Dinophysis species collected from Japanese coastal waters. A total of 715 nucleomorph sequences obtained from 134 M. rubra cells and 564 plastid 16S rRNA gene and 355 psbA sequences from 71 Dinophysis cells were determined. Almost all sequences in M. rubra and Dinophysis spp. were identical to those of Teleaulax amphioxeia, suggesting that M. rubra in Japanese coastal waters preferentially ingest T. amphioxeia. The remaining sequences were closely related to those of Geminigera cryophila and Teleaulax acuta. Interestingly, 37 plastid 16S rRNA gene sequences, which were different from T. amphioxeia and amplified from Dinophysis acuminata and Dinophysis norvegica cells, were identical to the sequence of a D. acuminata cell found in the Greenland Sea, suggesting that a widely distributed and unknown cryptophyte species is also preyed upon by M. rubra and subsequently sequestered by Dinophysis. To confirm the reliability of molecular identification of the cryptophyte Teleaulax species detected from M. rubra and Dinophysis cells, the nucleomorph and plastid genes of Teleaulax species isolated from seawaters were also analyzed. Of 19 isolates, 16 and 3 clonal strains were identified as T. amphioxeia and T. acuta, respectively, and no sequence variation was confirmed within species. T. amphioxeia is probably the primary source of prey for M. rubra in Japanese coastal waters. An unknown cryptophyte may serve as an additional source, depending on localities and seasons. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


PubMed | Japan National Research Institute of Fisheries Science, National Institute of Genetics, Japan National Research Institute of Fisheries And Environment of Inland Sea, Kumamoto Prefectural Fisheries Research Center and 2 more.
Type: Journal Article | Journal: Gene | Year: 2015

In this study, we investigated the influence of diurnal sampling bias on the community structure of plankton by comparing the biodiversity among seawater samples (n=9) obtained every 3h for 24h by using massively parallel sequencing (MPS)-based plankton monitoring at a fixed point conducted at Himedo seaport in Yatsushiro Sea, Japan. The number of raw operational taxonomy units (OTUs) and OTUs after re-sampling was 507-658 (558 104, mean standard deviation) and 448-544 (467 81), respectively, indicating high plankton biodiversity at the sampling location. The relative abundance of the top 20 OTUs in the samples from Himedo seaport was 48.8-67.7% (58.0 5.8%), and the highest-ranked OTU was Pseudo-nitzschia species (Bacillariophyta) with a relative abundance of 17.3-39.2%, followed by Oithona sp. 1 and Oithona sp. 2 (Arthropoda). During seawater sampling, the semidiurnal tidal current having an amplitude of 0.3ms(-1) was dominant, and the westward residual current driven by the northeasterly wind was continuously observed during the 24-h monitoring. Therefore, the relative abundance of plankton species apparently fluctuated among the samples, but no significant difference was noted according to G-test (p>0.05). Significant differences were observed between the samples obtained from a different locality (Kusuura in Yatsushiro Sea) and at different dates, suggesting that the influence of diurnal sampling bias on plankton diversity, determined using the MPS-based survey, was not significant and acceptable.


Shikata T.,Prefectural University of Kumamoto | Shikata T.,Graduate University for Advanced Studies | Sakurada K.,Kumamoto prefectural Fisheries Research Center | Jomoto Y.,Prefectural University of Kumamoto | And 4 more authors.
Nippon Suisan Gakkaishi (Japanese Edition) | Year: 2010

In the Yatsushiro Sea, we investigated the population dynamics of phytoplankton and some environmental factors, such as water temperature, salinity and underwater visibility. The field investigations at 8 offshorestations and Himedo were conducted monthly from April 2008 to March 2009 and weekly from June to October 2008, respectively. Moreover, we examined the growth of main phytoplankton species under different conditions of temperature, salinity and light intensity in the laboratory. Diatoms such as Skeletonerna costa turn, Chaetoceros spp., Thalassiosira spp. and Asterionellopsis gracialis dominated throughout the investigation periods, except for a Chattonella antiqua bloom in August 2008 in the sea. However, the dominant species of diatom fluctuated over time. Laboratory studies indicated that the growth characteristics of main phytoplankton species under different condi tions of water temperature and salinity vary among species, where as the growth characteristics under different light intensities are similar to each other; the growth of most phytoplankton species tested saturated at a light intensity of 80 mo-2 ms-1 . From statistical analyses with laboratory data, we found that the seasonal and temporal dynamics of dominant phytoplankton species are closely related to water temperature and salinity and underwater light intensity, respectively, in the Yatsushiro Sea.


Nishitani G.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Nishitani G.,Tohoku University | Nagai S.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Hayakawa S.,National Institute of Genetics | And 4 more authors.
Applied and Environmental Microbiology | Year: 2012

Kleptoplastidy is the retention of plastids obtained from ingested algal prey, which may remain temporarily functional and be used for photosynthesis by the predator. We showed that the marine dinoflagellate Dinophysis mitra has great kleptoplastid diversity. We obtained 308 plastid rbcL sequences by gene cloning from 14 D. mitra cells and 102 operational taxonomic units (OTUs). Most sequences were new in the genetic database and positioned within Haptophyceae (227 sequences [73.7%], 80 OTUs [78.4%]), particularly within the genus Chrysochromulina. Others were closely related to Prasinophyceae (16 sequences [5.2%], 5 OTUs [4.9%]), Dictyochophyceae (14 sequences [4.5%], 5 OTUs [4.9%]), Pelagophyceae (14 sequences [4.5%], 1 OTU [1.0%]), Bolidophyceae (3 sequences [1.0%], 1 OTU [1.0%]), and Bacillariophyceae (1 sequence [0.3%], 1 OTU [1.0%]); however, 33 sequences (10.8%) as 9 OTUs (8.8%) were not closely clustered with any particular group. Only six sequences were identical to those of Chrysochromulina simplex, Chrysochromulina hirta, Chrysochromulina sp. TKB8936, Micromonas pusilla NEPCC29, Micromonas pusilla CCMP491, and an unidentified diatom. Thus, we detected >100 different plastid sequences from 14 D. mitra cells, strongly suggesting kleptoplastidy and the need for mixotrophic prey such as Laboea, Tontonia, and Strombidium-like ciliates, which retain numerous symbiotic plastids from different origins, for propagation and plastid sequestration. © 2012, American Society for Microbiology.


Aoki K.,Japan National Research Institute of Fisheries Science | Onitsuka G.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Shimizu M.,Japan National Research Institute of Fisheries Science | Kuroda H.,Hokkaido National Fisheries Research Institute | And 6 more authors.
Marine Pollution Bulletin | Year: 2014

The dynamics of river plume in relation to harmful blooms of the raphidophycean flagellate, Chattonella antiqua in summer 2008-2010 in the Yatsushiro Sea, Japan were studied using a hydrodynamic model and monitoring data. In the southern area, the bloom formed in the waters stratified by a halocline caused by the southward expansion of riverine water from the Kuma River after the bloom initially forming in the northern area. The timing of the southward riverine water advection can be explained by the balance between the wind stress term and the pressure gradient term calculated from the horizontal density difference between the northern and southern areas. The wind stress and pressure gradient terms were evaluated using the sea surface temperature, salinity, wind speed and direction at two stations. Real time monitoring or continuous observations in these areas will enable nowcasts of bloom expansion when a bloom develops in the northern area. © 2014 Elsevier Ltd.


PubMed | Japan National Research Institute of Fisheries Science, Kagoshima Prefectural Fisheries Technology and Development Center, Japan National Research Institute of Fisheries And Environment of Inland Sea, Kumamoto Prefectural Fisheries Research Center and 2 more.
Type: Journal Article | Journal: Marine pollution bulletin | Year: 2014

The dynamics of river plume in relation to harmful blooms of the raphidophycean flagellate, Chattonella antiqua in summer 2008-2010 in the Yatsushiro Sea, Japan were studied using a hydrodynamic model and monitoring data. In the southern area, the bloom formed in the waters stratified by a halocline caused by the southward expansion of riverine water from the Kuma River after the bloom initially forming in the northern area. The timing of the southward riverine water advection can be explained by the balance between the wind stress term and the pressure gradient term calculated from the horizontal density difference between the northern and southern areas. The wind stress and pressure gradient terms were evaluated using the sea surface temperature, salinity, wind speed and direction at two stations. Real time monitoring or continuous observations in these areas will enable nowcasts of bloom expansion when a bloom develops in the northern area.


Nagai S.,Japan National Research Institute of Fisheries Science | Miyamoto S.,Kaneka Corporation | Ino K.,Kaneka Corporation | Tajimi S.,Kumamoto Prefectural Fisheries Research Center | And 2 more authors.
Harmful Algae | Year: 2016

In this study, the Kaneka DNA chromatography chip (KDCC) for the Alexandrium species was successfully developed for simultaneous detection of five Alexandrium species. This method utilizes a DNA-DNA hybridization technology. In the PCR process, specifically designed tagged-primers are used, i.e. a forward primer consisting of a tag domain, which can conjugate with gold nanocolloids on the chip, and a primer domain, which can anneal/amplify the target sequence. However, the reverse primer consists of a tag domain, which can hybridize to the solid-phased capture probe on the chip, and a primer domain, which can anneal/amplify the target sequence. As a result, a red line that originates from gold nanocolloids appears as a positive signal on the chip, and the amplicon is detected visually by the naked eye. This technique is simple, because it is possible to visually detect the target species soon after (<5 min) the application of 2 μL of PCR amplicon and 65 μL of development buffer to the sample pad of the chip. Further, this technique is relatively inexpensive and does not require expensive laboratory equipment, such as real-time Q-PCR machines or DNA microarray detectors, but a thermal cycler. Regarding the detection limit of KDCC for the five Alexandrium species, it varied among species and it was <0.1-10 pg and equivalent to 5-500 copies of rRNA genes, indicating that the technique is sensitive enough for practical use to detect several cells of the target species from 1 L of seawater. The detection sensitivity of KDCC was also evaluated with two different techniques, i.e. a multiplex-PCR and a digital DNA hybridization by digital DNA chip analyzer (DDCA), using natural plankton assemblages. There was no significant difference in the detection sensitivity among the three techniques, suggesting KDCC can be readily used to monitor the HAB species. © 2015 Elsevier B.V.


Tomaru Y.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Toyoda K.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Toyoda K.,Keio University | Kimura K.,Japan National Research Institute of Fisheries And Environment of Inland Sea | And 4 more authors.
Phycological Research | Year: 2013

Diatoms are the major primary producers in the world's aquatic environment; hence, their dynamics are an important focus in current studies. Viruses, along with other physical, chemical, and biological factors, have recently been recognized as potential factors of diatom mortality. We isolated and characterized a new diatom virus (Csp03RNAV) that causes lysis of the marine planktonic diatom Chaetoceros sp. strain SS08-C03 isolated from Hiroshima Bay, Japan. Here, we present the physiology, morphology, and genome characteristics of this virus. Csp03RNAV was isolated from surface waters of Yatsushiro Sea, Japan. Virions were icosahedral and 32nm in diameter, and accumulated in the cytoplasm of the host cells. The latent period was estimated to be <48h. Csp03RNAV harbors a single-stranded RNA genome, which has 9417 bases encoding two open reading frames that code for putative replication-related proteins and putative structural proteins, respectively. The monophyly of Csp03RNAV and the other known diatom-infecting single-stranded RNA viruses (genus Bacillarnavirus), Rhizosolenia setigera RNA virus, Chaetoceros socialis f. radians RNA virus, and Chaetoceros tenuissimus RNA virus was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domain. On the basis of these results, Csp03RNAV is considered to be a new member of the genus Bacillarnavirus. © 2012 Japanese Society of Phycology.


PubMed | Japan National Research Institute of Fisheries Science, Kagoshima Prefectural Fisheries Technology and Development Center, Kumamoto Prefectural Fisheries Research Center and Kaneka Corporation
Type: | Journal: Harmful algae | Year: 2016

In this study, the Kaneka DNA chromatography chip (KDCC) for the Alexandrium species was successfully developed for simultaneous detection of five Alexandrium species. This method utilizes a DNA-DNA hybridization technology. In the PCR process, specifically designed tagged-primers are used, i.e. a forward primer consisting of a tag domain, which can conjugate with gold nanocolloids on the chip, and a primer domain, which can anneal/amplify the target sequence. However, the reverse primer consists of a tag domain, which can hybridize to the solid-phased capture probe on the chip, and a primer domain, which can anneal/amplify the target sequence. As a result, a red line that originates from gold nanocolloids appears as a positive signal on the chip, and the amplicon is detected visually by the naked eye. This technique is simple, because it is possible to visually detect the target species soon after (<5min) the application of 2L of PCR amplicon and 65L of development buffer to the sample pad of the chip. Further, this technique is relatively inexpensive and does not require expensive laboratory equipment, such as real-time Q-PCR machines or DNA microarray detectors, but a thermal cycler. Regarding the detection limit of KDCC for the five Alexandrium species, it varied among species and it was <0.1-10pg and equivalent to 5-500 copies of rRNA genes, indicating that the technique is sensitive enough for practical use to detect several cells of the target species from 1L of seawater. The detection sensitivity of KDCC was also evaluated with two different techniques, i.e. a multiplex-PCR and a digital DNA hybridization by digital DNA chip analyzer (DDCA), using natural plankton assemblages. There was no significant difference in the detection sensitivity among the three techniques, suggesting KDCC can be readily used to monitor the HAB species.

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