Horizontal and vertical distribution of Gambierdiscus spp. (Dinophyceae) including novel phylotypes in Japan identified by 18S rDNA metabarcoding.

The genus Gambierdiscus is a marine benthic/epiphytic dinoflagellate considered the causative agent of ciguatera poisoning (CP). Clarifying the geographical distribution of this genus to understand the potential risk of CP is important. Many studies have focused only on the species/phylotype composition of Gambierdiscus in shallow waters, but no study has investigated the species/phylotype composition of the genus in deep waters. In the present study, the distributions of Gambierdiscus species/phylotypes at two depths (2-8 and 30 m) and two sampling sites (temperate and subtropical) in Japan was investigated using high throughput sequencing (HTS) with a newly developed primer set that preferentially amplifies the 18S rDNA V8-V9 region of Alveolata. A phylogenetic analysis using 89 samples collected over three years revealed of ten Gambierdiscus species/phylotypes including not only two species that have not been reported in Japan (G. caribaeus and G. silvae) but also four novel phylotypes (Gambierdiscus spp. Clade II_1, Clade II_2, Clade II_3, and Clade VI_1). Uncorrected genetic distances also supported that these new phylotypes clearly diverged from other Gambierdiscus species. All four new phylotypes, G. caribaeus, and G. silvae were distributed in the subtropical region. Among them, Clade II_2, Clade VI_1, and G. silvae were also distributed in the temperate region. Four species/phylotypes previously reported from Japan showed a similar distribution as reported previously. Among the ten species/phylotypes, Gambierdiscus sp. type 3 and Clade VI_1 were found only in deep waters, whereas five species/phylotypes were observed only in shallow waters. The other three species/phylotypes were found in both deep and shallow waters. The results of the horizontal and vertical distribution suggest that the growth characteristics of each species/phylotypes found in Japan might adapt to the ambient environmental conditions. This study revealed an inclusive assemblage of Gambierdiscus species/phylotypes in Japan through metabarcoding using the Alveolata primer set. In the future, the abundance and toxicities/toxin productions of the newly reported species/phylotypes need to be clarified to understand the mechanism of CP outbreaks in Japan.

pH-Activatable Cyanine Dyes for Selective Tumor Imaging Using Near-Infrared Fluorescence and Photoacoustic Modalities.

Photoacoustic (PA) imaging is an emerging molecular imaging modality that complements fluorescence imaging and enables high resolution within deep tissue. Fluorescence/PA multimodality imaging would be a powerful technique to extract more comprehensive information from targets than traditional single-modality imaging. In this paper, we developed a new pH-activatable sensor, CypHRGD, which is applicable to both fluorescence and PA detection. CypHRGD was derived from our previous near-infrared pH-sensing platform, in which substitution with a bulky phenyl group and functionalization with a cRGD peptide remarkably improved the sensor's biocompatibility with attenuated dye aggregation. The multimodality imaging applications of CypHRGD were demonstrated in cultured cells and cancer-xenografted mice with rapid kinetics and high sensitivity and specificity, which relies on cRGD-facilitated tumor targeting, probe accumulation and subsequent activation in the acidic organelles after endocytosis.

First report of Alexandrium (Dinophyceae) associated with marine macroalgae off Japan: Diversity, distribution, and toxicity.

Macroalgal samples were collected from coastal waters in subboreal to subtropical zones in Japan (< 3-30 m depths) and 32 clonal strains of non-motile dinoflagellate-like protists were established. Molecular phylogenetic analyses of the LSU rDNA D1/D2, SSU rDNA, ITS region, and concatenated SSU rDNA + LSU rDNA D1/D2 sequences revealed that the strains nested within the genus Alexandrium. They were separated into three novel phylotypes: Alexandrium spp. type 1, type 2, and type 3. Analysis of the concatenated sequences revealed that the most closely related species for the three phylotypes was A. ostenfeldii. Most cells from strains of the three phylotypes were non-motile and hemispherical to spherical in shape. The average diameters of the non-motile cells were between 35 and 39 µm. Type 1 and type 2 were widely distributed in Japan from the temperate to subtropical zones, whereas type 3 was restricted to the temperate zone. Furthermore, type 2 was widespread from shallow to deep waters, whereas type 1 and type 3 were restricted to deep waters. Growth experiments in strains belonging to the three phylotypes revealed that the occurrence ratios of motile cells were very low (≤ 1.1% of the total cells). The production of paralytic shellfish poisoning toxins, tetrodotoxin, and cyclic imines was assessed in strains belonging to the three phylotypes by LC/MS/MS analysis. The strains did not produce any of the toxins tested. The strains of the three phylotypes showed lethal toxicity to mice by intraperitoneal administration. To the best of our knowledge, this is the first study to report the existence of Alexandrium associated with marine macroalgae from Japan.

Chronological distribution of dinoflagellate-infecting RNA virus in marine sediment core.

A bivalve-killing marine dinoflagellate, Heterocapsa circularisquama, is susceptible to the infectious single-stranded RNA virus, Heterocapsa circularisquama RNA virus (HcRNAV). The ecological relationship between H. circularisquama and HcRNAV was intensively studied from 2001 through 2005; however, only limited data are available for the ecological dynamics of HcRNAV before 2001. In this study, we applied radiometric dating and reverse transcription PCR (RT-PCR) to determine the chronological distribution of HcRNAV in a marine sediment core sampled from the Uranouchi Inlet, Kochi, Japan, where H. circularisquama was first discovered. Our results show that HcRNAV had existed in the inlet long before its first bloom in 1988. Furthermore, five HcRNAV variants, phylogenetically distinguishable based on the nucleotide sequence of the major capsid protein (MCP) gene, were identified. These variants were found to be distributed throughout the core over time, suggesting that the HcRNAV sequences registered in the NCBI database are only a portion of the variants that have emerged in the history of HcRNAV diversification. Herein, we have verified the applicability of the retrospective approach for speculating the distribution of algal RNA viruses over time in aquatic environments.

Abundance of the benthic dinoflagellate Prorocentrum and the diversity, distribution, and diarrhetic shellfish toxin production of Prorocentrum lima complex and P. caipirignum in Japan.

In the present study, the abundance of Prorocentrum and the molecular phylogeny, distribution, and DST production of P. lima complex and P. caipirignum in Japan were investigated. First, the cell densities of Prorocentrum were assessed from the temperate to subtropical zones in Japan between 2014 and 2018. The cell density in the subtropical zone [19.0 ± 40.2 cells/g wet weight (ww) algae] was significantly higher than that in the temperate zone (1.4 ± 3.4 cells/g ww algae). A total of 244 clonal strains were established from the temperate and subtropical zones. Phylogenetic analyses based on the large-subunit ribosomal DNA D1/D2 revealed that the strains were separated into four species/species complex/phylotypes (P. lima complex, P. caipirignum, and new phylotypes Prorocentrum spp. types 1 and 2). The strains of P. lima complex could be separated into two clades (1 and 3). Furthermore, the strains of clades 1 and 3 could be separated into nine subclades (1a, 1c, 1d, 1e, 1f, 1g, 1h, 1i, and 1j) and three subclades (3a, 3b, and 3c), respectively. The strains of P. caipirignum were separated into two subclades (b and e). Each phylotype/subclade showed a unique distribution pattern in Japan: P. lima complex subclades 1a, 1c, and 3a and P. caipirignum subclades b and e were widespread from the temperate to subtropical zones. On the other hand, P. lima complex subclades 1e and 1i were restricted to the temperate zone, and P. lima complex subclades 1d, 1f, 1g, 1h, 1j, 3b, and 3c and Prorocentrum spp. types 1 and 2 were restricted to the subtropical zone. Furthermore, the DST production of the 243 clonal strains was assessed by LC/MS/MS analysis. The results revealed that all strains produced okadaic acid (OA) and that the OA contents of P. lima complex subclades 1d and 1f, P. caipirignum subclades b and e, and Prorocentrum sp. type 2 tended to be higher than those of the other subclades. While P. lima complex subclades 1a, 1e, 1f, and 1i produced DTX1, the other phylotype/subclades produced either no or low quantities of DTX1. A strain of P. lima complex subclade 1e showed the highest OA and DTX1 contents (55.27 and 70.73 pg/cell, respectively) in the world. These results suggest that there are potential risks for DST accumulation in benthic animals in Japan.

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae).

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R>0.92 (p<0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (Lmax) and the semi-optimum light intensity range (Ls-opt) that resulted in the maximum growth rate (µmax) and ≥80% µmax values of the four species/phylotypes, respectively, were as follows: (1) the Lmax and Ls-opt of G. australes were 208µmol photons m-2s-1 and 91-422µmol photons m-2s-1, respectively; (2) those of G. scabrosus were 252 and 120-421µmol photons m-2s-1, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75-430µmol photons m-2s-1, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73-427µmol photons m-2s-1, respectively. All four Gambierdiscus species/phylotypes required approximately 10µmol photons m-2s-1 to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer's Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058-0.119m-1. The values 1700µmol photons m-2s-1, 500µmol photons m-2s-1, and 200µmol photons m-2s-1 were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of Lmax and Ls-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12-38m and 12-54m, 1-16m and 1-33m, and 0m and 0-16m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.

Simultaneous detection of multiple hydroxylated polychlorinated biphenyls from a complex tissue matrix using gas chromatography/isotope dilution mass spectrometry.

In this study, we developed a comprehensive, highly sensitive, and robust method for determining 53 congeners of three to eight chlorinated OH-PCBs in liver and brain samples by using isotope dilution gas chromatography (GC) coupled with electron capture negative ionization mass spectrometry (ECNI-MS). These results were compared with those from GC coupled with electron ionization high-resolution mass spectrometry (EI-HRMS). Clean-up procedures for analysis of OH-PCBs homologs in liver and brain samples involve a pretreatment step consisting of acetonitrile partition and 5% hydrated silica-gel chromatography before derivatization. Recovery rates of tri- and tetra-chlorinated OH-PCBs in the acetonitrile partition method followed by the 5% hydrated silica-gel column (82% and 91%) were higher than conventional sulfuric acid treatment (2.0% and 3.5%). The method detection limits of OH-PCBs for each matrix obtained by GC/ECNI-MS and GC/EI-HRMS were 0.58-2.6 pg g(-1) and 0.36-1.6 pg g(-1) wet wt, respectively. Recovery rates of OH-PCB congeners in spike tests using sample matrices (10 and 50 pg) were 64.7-117% (CV: 4.7-14%) and 70.4-120% (CV: 2.3-12%), respectively. This analytical method may enable the simultaneous detection of various OH-PCBs from complex tissue matrices. Furthermore, this method allows more comprehensive assessment of the biological effects of OH-PCB exposure on critical organs.

Coverage, diversity, and functionality of a high-latitude coral community (Tatsukushi, Shikoku Island, Japan).

BACKGROUND: Seawater temperature is the main factor restricting shallow-water zooxanthellate coral reefs to low latitudes. As temperatures increase, coral species and perhaps reefs may move into higher-latitude waters, increasing the chances of coral reef ecosystems surviving despite global warming. However, there is a growing need to understand the structure of these high-latitude coral communities in order to analyze their future dynamics and to detect any potential changes. METHODOLOGY/PRINCIPAL FINDINGS: The high-latitude (32.75°N) community surveyed was located at Tatsukushi, Shikoku Island, Japan. Coral cover was 60±2% and was composed of 73 scleractinian species partitioned into 7 functional groups. Although only 6% of species belonged to the 'plate-like' functional group, it was the major contributor to species coverage. This was explained by the dominance of plate-like species such as Acropora hyacinthus and A. solitaryensis. Comparison with historical data suggests a relatively recent colonization/development of A. hyacinthus in this region and a potential increase in coral diversity over the last century. Low coverage of macroalgae (2% of the benthic cover) contrasted with the low abundance of herbivorous fishes, but may be reasonably explained by the high density of sea urchins (12.9±3.3 individuals m⁻²). CONCLUSIONS/SIGNIFICANCE: The structure and composition of this benthic community are relatively remarkable for a site where winter temperature can durably fall below the accepted limit for coral reef development. Despite limited functionalities and functional redundancy, the current benthic structure might provide a base upon which a reef could eventually develop, as characterized by opportunistic and pioneer frame-building species. In addition to increasing seawater temperatures, on-going management actions and sea urchin density might also explain the observed state of this community. A focus on such 'marginal' communities should be a priority, as they can provide important insights into how tropical corals might cope with environmental changes.

Warming off southwestern Japan linked to distributional shifts of subtidal canopy-forming seaweeds.

To assess distributional shifts of species in response to recent warming, historical distribution records are the most requisite information. The surface seawater temperature (SST) of Kochi Prefecture, southwestern Japan on the western North Pacific, has significantly risen, being warmed by the Kuroshio Current. Past distributional records of subtidal canopy-forming seaweeds (Laminariales and Fucales) exist at about 10-year intervals from the 1970s, along with detailed SST datasets at several sites along Kochi's >700 km coastline. In order to provide a clear picture of distributional shifts of coastal marine organisms in response to warming SST, we observed the present distribution of seaweeds and analyzed the SST datasets to estimate spatiotemporal SST trends in this coastal region. We present a large increase of 0.3°C/decade in the annual mean SST of this area over the past 40 years. Furthermore, a comparison of the previous and present distributions clearly showed the contraction of temperate species' distributional ranges and expansion of tropical species' distributional ranges in the seaweeds. Although the main temperate kelp Ecklonia (Laminariales) had expanded their distribution during periods of cooler SST, they subsequently declined as the SST warmed. Notably, the warmest SST of the 1997-98 El Niño Southern Oscillation event was the most likely cause of a widespread destruction of the kelp populations; no recovery was found even in the present survey at the formerly habitable sites where warm SSTs have been maintained. Temperate Sargassum spp. (Fucales) that dominated widely in the 1970s also declined in accordance with recent warming SSTs. In contrast, the tropical species, S. ilicifolium, has gradually expanded its distribution to become the most conspicuously dominant among the present observations. Thermal gradients, mainly driven by the warming Kuroshio Current, are presented as an explanation for the successive changes in both temperate and tropical species' distributions.

[Denaturation of egg antigens by cooking].

Changes in egg protein contents by cooking were measured with an ELISA kit using Tris-HCl buffer in model foods including cake, meatballs, pasta and pudding made with whole egg, egg-white and egg-yolk. The egg protein contents were lowest in the deep-fried model foods of cakes and meatballs. Ovalbumin (OVA) was undetectable (<1 µg/g) and ovomucoid (OVM) was lowest in pouched meatballs, suggesting that processing temperature and uniform heat-treatment affect the detection of egg protein. Furthermore, egg protein contents were below 6 µg/g in the pouched meatballs and pasta made with egg-yolk, and OVA and OVM were not detected by Western blotting analysis with human IgE from patients' serum. On the other hand, processed egg proteins were detected with an ELISA kit using a surfactant and reductant in the extract buffer.

[Comparison between old and new methods for detection of allergenic substances (egg and milk)].

The old ELISA method for detection of allergenic substances (egg and milk) in Kanagawa prefecture from 2003 to 2007, employed before improvement of the food allergen labeling system, yielded detection rates of 20% for egg and 30% for milk. In 2005, after improvement of the labeling system, the detection rate using the new ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol increased by about 10% for egg, but decreased by half for milk. There were 4 positive samples (over 10 µg/g) for both egg and milk proteins, on account of contamination by ingredients at the manufacturing line and the lack of proper food labeling. In 2009, the contamination levels of egg and milk proteins in labeled commercial foods were low. In a comparison between the new and old methods with the same samples, both the new ELISA and Western-blot analyses showed an increase in the detection rate of egg protein. In relation to milk protein, the detection rates were decreased with the new ELISA, although the ELISA detection rate and consistency rates with Western-blot analysis were increased. On the other hand, in the case of a protein content below 5 µg/g, it was impossible to determine ovomucoid and casein by Western-blot analysis.