Morphological variation and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an intensive cold-water algal bloom in eastern Hokkaido, Japan.

Harmful algal blooms responsible for mass mortalities of marine organisms have been rare in Hokkaido, northern Japan, although fish-killing blooms have been frequently reported from western Japanese coasts. In September-November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurring unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1-D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis, with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of Kr. selliformis in the bloom were discoid, dorsoventrally flattened, and 35.3-43.6 (39.4  ±  2.1) µm in length, which was larger than the cell sizes in previous reports. Transparent cells of Kr. selliformis, lacking chloroplasts or having a few shrunken chloroplasts and oil droplets, were also found. Cells of Kr. selliformis showed morphological variation, but the species could be distinguished from other co-occurring Karenia species by the nucleus positioned in the hypocone and chloroplasts numerous (46-105) in number and small (2.9-4.6 µm) in diameter. Cell density of Kr. selliformis exceeding 100 cells mL-1 was recorded in the temperature range of 9.8-17.6 °C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from the bloom in Kamchatka, Russia in 2020.

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes.

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•-) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•- production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•- production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.

The harmful raphidophyte Chattonella (Raphidophyceae) in Western Pacific: Its red tides and associated fisheries damage over the past 50 years (1969-2019).

Red tides and associated fisheries damage caused by the harmful raphidophyte Chattonella were reassessed based on the documented local records for 50 years to understand the distribution and economic impacts of the harmful species in the Western Pacific. Blooms of Chattonella with fisheries damage have been recorded in East Asia since 1969, whereas they have been only recorded in Southeast Asia since the 1980s. Occurrences of Chattonella have been documented from six Southeast Asian countries, Indonesia, Malaysia, Philippines, Singapore, Thailand and Viet Nam, with mass mortalities mainly of farmed shrimp in 1980-1990s, and farmed fish in 2000-2010s. These occurrences have been reported with the names of C. antiqua, C. marina, C. ovata, C. subsalsa and Chattonella sp., owing to the difficulty of microscopic species identification, and many were not supported with molecular data. To determine the distribution of C. marina complex and C. subsalsa in Southeast Asia, molecular phylogeny and microscopic observation were also carried out for cultures obtained from Indonesia, Malaysia, Japan, Philippines, Russia, Singapore and Thailand. The results revealed that only the genotype of C. marina complex has been detected from East Asia (China, Japan, Korea and Russia), whereas both C. marina complex (Indonesia and Malaysia) and C. subsalsa (Philippines, Singapore and Thailand) were found in Southeast Asia. Ejection of mucocysts has been recognized as a diagnostic character of C. subsalsa, but it was also observed in our cultures of C. marina isolated from Indonesia, Malaysia, Japan, and Russia. Meanwhile, the co-occurrences of the two harmful Chattonella species in Southeast Asia, which are difficult to distinguish solely based on their morphology, suggest the importance of molecular identification of Chattonella genotypes for further understanding of their distribution and negative impacts.

Harmful algal blooms and associated fisheries damage in East Asia: Current status and trends in China, Japan, Korea and Russia.

Occurrences of harmful algal blooms (HABs) and associated fisheries damage have been continuously monitored since the 1970s along the coasts of East Asia. Fisheries damage comprises mass mortalities of fish and shellfish mainly by harmful dinoflagellates and raphidophytes (e.g., Chattonella antiqua/marina, Cochlodinium polykrikoides and Karenia mikimotoi), and contamination of algal toxins in shellfish in particular Diarrhetic Shellfish Toxins by Dinophysis spp. and Paralytic Shellfish Toxins by Alexandrium spp. Shellfish mass mortalities due to Heterocapsa circularisquama in Hong Kong and western Japan, and fish kills by Karlodinium digitatum are unique incidents for this region, whereas C. antiqua/marina, C. polykrikoides and K. mikimotoi are common also in other regions. Time series data showed that the highest bloom numbers were recorded in 1980 (Japan), in 1998 (Korea) and in 2003 (China), followed by decreasing trends in these countries. These data suggest a shift in microalgal species composition, from dominance by diatoms to dinoflagellates after 1980s in Korea, and from diatoms to small haptophytes and cyanobacteria after 2013 in eastern Russia. HAB species composition and the changes were compared among countries, for better understanding on current status and trend of HAB species in East Asia.

Interannual variations in abundance and distribution of Chattonella cysts, and the relationship to population dynamics of vegetative cells in the Yatsushiro Sea, Japan.

The fish-killing raphidophytes Chattonella spp. have a resting cyst stage. To investigate the abundance and distribution of Chattonella cysts and determine their relationship to the population dynamics of vegetative cells, we conducted field observations from 2002 to 2017 in the Yatsushiro Sea, a semi-enclosed embayment in Japan, and analyzed the data including environmental conditions. Analysis of sediment sampled in the spring (mid-April to early June), shows that cysts are relatively abundant in the northern to middle area, where initial vegetative cells and large blooms are frequently detected. The maximum density of cysts was 616 cysts cm-3 in the northern area in 2016. Mean cyst abundance in the spring varied interannually, ranging from 5 to 138 cysts cm-3. A significant positive correlation between mean cyst abundance in the spring and maximum density of vegetative cells the preceding summer was seen, but no significant correlation was observed the following summer. The first detected date of vegetative cells (FDD) each year, which is likely related to cyst abundance and environmental conditions influencing cyst germination and/or growth characteristics of vegetative cells, also varied interannually from mid-April to early June. Regression analyses showed that FDD tended to be early when cyst abundance and bottom-water temperature were high. However, no significant correlation was observed between mean cyst abundance and bloom timing (the period from FDD to the occurrence date of the bloom), and bloom duration the following summer, as was the maximum density of vegetative cells. Instead, the timing and duration of blooms were correlated significantly with meteorological factors (e.g., solar radiation) for a month after FDD. The results suggest that cyst abundance reflecting the bloom magnitude of the preceding summer contributes to the timing of the appearance of vegetative cells in the year, but that bloom occurrence is likely to be controlled by the growth dynamics of vegetative cells through environmental conditions rather than by cyst abundance. The three distinct peaks in Chattonella cysts and vegetative cells from 2002 to 2017 correspond to the timings just after the El Niño. Large-scale atmospheric variability and its global teleconnection are possibly linked to long-term population dynamics of Chattonella in the area through local meteorological conditions and their life cycle.

RNA-Seq Analysis Reveals Genes Related to Photoreception, Nutrient Uptake, and Toxicity in a Noxious Red-Tide Raphidophyte Chattonella antiqua.

Aquaculture industries are under threat from noxious red tides, but harm can be mitigated by precautions such as early harvesting and restricting fish feeding to just before the outbreak of a red tide. Therefore, accurate techniques for forecasting red-tide outbreaks are strongly needed. Omics analyses have the potential to expand our understanding of the eco-physiology of these organisms at the molecular level, and to facilitate identification of molecular markers for forecasting their population dynamics and occurrence of damages to fisheries. Red tides of marine raphidophytes, especially Chattonella species, often extensively harm aquaculture industries in regions with a temperate climate around the world. A red tide of Chattonella tends to develop just after an input of nutrients along the coast. Chattonella displays diurnal vertical migration regulated by a weak blue light, so it photosynthesizes in the surface layer during the daytime and takes up nutrients in the bottom layer during the nighttime. Superoxide produced by Chattonella cells is a strong candidate for the cause of its toxicity to bacteria and fishes. Here we conducted mRNA-seq of Chattonella antiqua to identify genes with functions closely related to the dynamics of the noxious red tide, such as photosynthesis, photoreception, nutrient uptake, and superoxide production. The genes related to photosynthetic pigment biosynthesis and nutrient uptake had high similarity with those of model organisms of plants and algae and other red-tide microalgae. We identified orthologous genes of photoreceptors such as aureochrome (newly five genes), the cryptochrome/photolyase (CRY/PHR) family (6-4PHR, plant CRY or cyclobutane pyrimidine dimer [CPD] Class III, CPD Class II, and CRY-DASH), and phytochrome (four genes), which regulate various physiological processes such as flagellar motion and cell cycle in model organisms. Six orthologous genes of NADPH oxidase, which produces superoxide on the cell membrane, were found and divided into two types: one with 5-6 transmembrane domains and another with 11 transmembrane domains. The present study should open the way for analyzing the eco-physiological features of marine raphidophytes at the molecular level.

The Japanese Lung Cancer Society Guideline for non-small cell lung cancer, stage IV.

According to rapid development of chemotherapy in advanced non-small cell lung cancer (NSCLC), the Japan Lung Cancer Society has been updated its own guideline annually since 2010. In this latest version, all of the procedure was carried out in accordance with grading of recommendations assessment, development and evaluation (GRADE) system. It includes comprehensive literature search, systematic review, and determination of the recommendation by multidisciplinary expert panel which consisted of medical doctors, pharmacists, nurses, statisticians, and patients from patient advocacy group. Recently, we have had various types of chemotherapeutic drugs like kinase inhibitors or immune-checkpoint inhibitors. Thus, the guideline proposes to categorize patients into three entities: (1) driver oncogene-positive, (2) PD-L1 ≥ 50%, and (3) others. Based on this subgroup, 31 clinical questions were described. We believe that this attempt enables clinicians to choose appropriate treatment easier. Here, we report an English version of the Japan Lung Cancer Society Guidelines 2018 for NSCLC, stages IV.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates.

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Occurrence of Karenia papilionacea (Dinophyceae) and its novel sister phylotype in Japanese coastal waters.

Several species of the genus Karenia (Dinophyceae) form blooms and often cause the mortality of cultured and wild fish. In Japan, blooms caused by two species - namely Karenia mikimotoi and Karenia brevis - have been reported so far. On the basis of morphological and molecular-phylogenic examinations, the present investigation found Karenia papilionacea and its novel sister phylotype for the first time in the coastal waters of the various regions of Japan. Of 34 strains isolated from the coastal waters, 27 strains displayed the typical morphological characteristics of K. papilionacea and further showed consensus DNA sequences corresponding to those of the originally described K. papilionacea. The other 7 strains displayed the same morphological characteristics of K. papilionacea, but showed divergent DNA sequences, at a genetic distance of over 0.04 (Internal Transcribed Spacer regions) from those of the original phylotype of K. papilionacea. These divergent strains were characterized as a novel sister phylotype (phylotype-I) of K. papilionacea. In the coastal waters of Japan, K. papilionacea-like (K. papilionacea and/or its phylotype-I) formed blooms at 20.3-30.4°C and salinity 30.1-33.9. No K. brevis was identified in Japanese coastal waters during this study. These findings demonstrated that K. papilionacea occurs along the coasts of western Japan and possibly shares several coastal regions with K. mikimotoi and K. papilionacea phylotype-I. In order to assess the risks of Karenia blooms to aquaculture, it is essential that the growth physiology and ichthyotoxicity of K. papilionacea and its novel phylotype should be characterized.

Median-radial sensory nerve comparative studies in the detection of median neuropathy at the wrist in diabetic patients.

OBJECTIVE: Median-ulnar comparative studies (MUCS) play an important role in the electrodiagnosis of carpal tunnel syndrome, but in diabetes concomitant involvement of Guyon's canal (ulnar nerve compression at the wrist) would reduce the sensitivity of MUCS. This study tested the utility of median-radial comparative studies (MRCS) in diabetic patients. METHODS: Anti-dromic MUCS and MRCS were prospectively performed in 120 patients with diabetes, and 64 normal controls. In 28 diabetic patients, latent addition using threshold tracking was performed in superficial radial sensory axons to estimate persistent nodal sodium currents. RESULTS: MUCS was abnormal in 49% of the diabetic patients, and MRCS was abnormal in 58%. Median motor distal latencies were prolonged in 38%, and median sensory nerve conduction velocities were slowed in 40%. The longer latency differences in MRCS were associated with smaller persistent sodium currents, suggesting that intra-axonal sodium accumulation mediated by hyperglycemia enhances nerve compression. CONCLUSIONS: MRCS appears to be the most sensitive electrodiagnostic test in the detection of median neuropathy at the wrist in diabetic patients. Nerve conduction slowing across the carpal tunnel may be associated with metabolic abnormalities under hyperglycemia. SIGNIFICANCE: Assessment of nerve conduction across the common entrapment sites could provide new insights into the pathophysiology of diabetic neuropathy related to metabolic factors.

Remarkable difference in accumulation of paralytic shellfish poisoning toxins among bivalve species exposed to Pyrodinium bahamense var. compressum bloom in Masinloc Bay, Philippines.

Seasonal variation of bivalve toxicity was monitored in association with the abundance of the toxic dinoflagellate Pyrodinium bahamense var. compressum in Masinloc Bay, Luzon Island. Among 7 species of bivalve, 6 species became toxic during a bloom of the dinoflagellate. However, remarkable difference in the toxicity was observed among the species. The toxicity of thorny oyster Spondylus squamosus was the highest among the species, showing more than 30 times that of safety consumption level after the peak bloom of the dinoflagellate, while other bivalve species showed much lower toxicity. The toxicity of thorny oyster decreased under absence of the dinoflagellate, but this species maintained a considerably high toxicity throughout a year. Similar trend was observed in penshell Atrina vexillum in a small scale, indicating that these species in the bay are not safe for human consumption almost throughout a year. The toxicity of green mussel Perna viridis increased to significant level during a bloom, but it decreased rapidly when the dinoflagellate disappeared. Toxin analysis of cultured and natural cells showed typical toxin profile of the dinoflagellate. Toxin profile of all the bivalve species reflected the characteristic toxin profile of the dinoflagellate.