New azaspiracid analogues detected as bi-charged ions in Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters.

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

Description of two new species Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov. (Raphidophyceae) from South China Sea, with a report of wild fish mortality.

Fisheries damage caused by Chattonella red tide has been recorded in Southeast Asia. Molecular studies have clarified the presence of two species, Chattonella marina complex and Chattonella subsalsa in the region, unlike East Asia that had only C. marina complex. To elucidate the phylogeography of Chattonella in Asia, further phylogenetic and morphological examinations were carried out with 33 additional culture strains, including the strains isolated during a bloom of Chattonella sp. (up to 142 cells mL-1) that was associated with a wild fish mortality along the northeastern coast of Peninsular Malaysia in 2016, and those from Yellow Sea, where the Chattonella genotypes have not been determined. LSU rDNA and ITS2 trees showed five intrageneric clades in the genus Chattonella, which were clades I and II (C. subsalsa), clade III (C. marina complex) and two new clades, namely clade IV from Thailand and Malaysia, and clade V from Peninsular Malaysia. The positions of the two new clades were different in LSU rDNA and ITS2 trees. LSU rDNA divergences of clades IV and V from the other clades were ≥ 4.01% and ≥ 5.70%, while their ITS2 divergences were ≥ 7.44% and ≥ 16.43%, respectively. Three and five compensatory base changes (CBCs) were observed in the clades IV and V, respectively, when compared to each of their closest clade. Cells from clades IV and V showed similar morphology to C. marina complex and C. subsalsa clade II, including the presence of button-like granules on cell surface and oboe-shaped mucocysts. However, cell size, the number and shape of chloroplasts in Chattonella clades IV and V, and the non-stacked thylakoids penetrated the pyrenoid in C. subsalsa clade II, were distinctive. Based on the diagnostic chloroplast shape, we proposed the designation of clades IV and V to two new species, Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov.

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.

Thecal tabulation, body scale morphology and phylogeny of Heterocapsa philippinensis sp. nov. (Peridiniales, Dinophyceae) from the Philippines.

The thecal tabulation and body scale structure of the marine armoured dinoflagellate Heterocapsa, isolated from Philippines, were examined using LM, SEM and TEM, and its phylogenetic position was inferred from ITS and LSU rDNA sequences. Cells were ovoid and the plate tabulation (Po, cp, X, 5', 3a, 7'', 6c, 5s, 5''', 2'''') was consistent with most Heterocapsa species. The second anterior intercalary plate (2a) had a circular pattern with a thick marginal border free of pores. The nucleus was longitudinally elongated and curved, and located at the dorsal side of the cell. Discoid lobes of brownish chloroplast were peripherally distributed, and a pyrenoid was positioned at the centre. The triradiate body scales, measuring 250-300 nm in diameter, consisted of a roundish basal plate with six radiating ridges, nine peripheral uprights/spines, and three radiating spines. These components were identical to those of H. pseudotriquetra and H. steinii, except for the roundish outline of basal plate. Molecular phylogeny showed that the species clustered with H. pseudotriquetra and H. steinii. This species was differentiated from all other Heterocapsa species in the sausage-shaped nucleus and circular pattern on the 2a plate. This study proposed a novel species Heterocapsa philippinensis sp. nov. for the isolate.

Over 30 years of HABs in the Philippines and Malaysia: What have we learned?

In the Southeast Asian region, the Philippines and Malaysia are two of the most affected by Harmful Algal Blooms (HABs). Using long-term observations of HAB events, we determined if these are increasing in frequency and duration, and expanding across space in each country. Blooms of Paralytic Shellfish Toxin (PST)-producing species in the Philippines did increase in frequency and duration during the early to mid-1990s, but have stabilized since then. However, the number of sites affected by these blooms continue to expand though at a slower rate than in the 1990s. Furthermore, the type of HABs and causative species have diversified for both toxic blooms and fish kill events. In contrast, Malaysia showed no increasing trend in the frequency of toxic blooms over the past three decades since Pyrodinium bahamense was reported in 1976. However, similar to the Philippines, other PST producers such as Alexandrium minutum and Alexandrium tamiyavanichii have become a concern. No amnesic shellfish poisoning (ASP) has been confirmed in either Philippines or Malaysia thus far, while ciguatera fish poisoning cases are known from the Philippines and Malaysia but the causative organisms remain poorly studied. Since the 1990s and early 2000s, recognition of the distribution of other PST-producing species such as species of Alexandrium and Gymnodinium catenatum in Southeast Asia has grown, though there has been no significant expansion in the known distributions within the last decade. A major more recent problem in the two countries and for Southeast Asia in general are the frequent fish-killing algal blooms of various species such as Prorocentrum cordatum, Margalefidinium polykrikoides, Chattonella spp., and unarmored dinoflagellates (e.g., Karlodinium australe and Takayama sp.). These new sites affected and the increase in types of HABs and causative species could be attributed to various factors such as introduction through mariculture and eutrophication, and partly because of increased scientific awareness. These connections still need to be more concretely investigated. The link to the El Niño Southern Oscillation (ENSO) should also be better understood if we want to discern how climate change plays a role in these patterns of HAB occurrences.

The Harmful Unarmored Dinoflagellate Karlodinium in Japan and Philippines, with Reference to Ultrastructure and Micropredation of Karlodinium azanzae sp. nov. (Kareniaceae, Dinophyceae)1.

In all, 26 cultures of the harmful marine dinoflagellate Karlodinium, isolated from Japanese and Philippine coastal waters, were examined using LM, SEM, and molecular phylogeny inferred from ITS and LSU rDNA. Seven Karlodinium species (six from Japan and four from Philippines), K. australe, K. ballantinum, K. decipiens, K. gentienii, K. veneficum, K. zhouanum, and a novel species Karlodinium azanzae sp. nov., were identified based on their morphology and phylogenetic positions. Karlodinium azanzae from Manila Bay, Philippines was further characterized by TEM, HPLC (chloroplast pigment), and bioassay on brine shrimp and other marine zooplankton. Cells of K. azanzae were the largest (mean 25.3 µm long) in Karlodinium, possessed numerous tiny reflective particles, starch grains, and lipid granules, and usually swam at the bottom of the culture vessel. The straight apical structure complex and a ventral pore were common to the genus. The longitudinally elongated nucleus was located at the center, and the yellowish chloroplasts contained an embedded pyrenoid and carotenoid pigments typical of the genus (i.e., fucoxanthin as major carotenoid with its derivatives). TEM revealed a part of the flagellar apparatus, of which the long striated ventral connective is the first report in the Kareniaceae. Phylogenetic trees showed closest affinity of K. azanzae with K. australe and K. armiger. The new species could be differentiated from related species by cell size, position of the nucleus, and characteristic swimming behavior. Lethality of K. azanzae to large zooplankton and micropredation using a developed peduncle was also observed.

Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot.

Marine unarmored dinoflagellates in the family Kareniaceae are known to possess chloroplasts of haptophyte origin, which contain fucoxanthin and its derivatives as major carotenoids, and lack peridinin. In the present study, the first species with the peridinin-type chloroplast in this family, Gertia stigmatica gen. et sp. nov., is described on the basis of ultrastructure, photosynthetic pigment composition, and molecular phylogeny inferred from nucleus- and chloroplast-encoded genes. Cells of G. stigmatica were small and harboring a chloroplast with an eyespot and two pyrenoids. The apical structure complex was straight, similar to Karenia and Karlodinium. Under transmission electron microscopy, the chloroplast was surrounded by two membranes, and the eyespot was composed of a single layer of osmiophilic globules (eyespot type A); this was never previously reported from the Kareniaceae. High performance liquid chromatography demonstrated the chloroplast contains peridinin, and neither fucoxanthin nor 19'-acyloxyfucoxanthins was identified. A phylogeny based on nucleus-encoded rDNAs suggested a position of G. stigmatica in the Kareniaceae, but not clustered within the previously described genera, i.e., Karenia, Karlodinium and Takayama. A phylogeny of chloroplast-encoded psbA, psbC and psbD indicated the chloroplast is of peridinin-type typical of dinoflagellates, but the most related species remains unclear.

Taxonomy and toxin production of Gambierdiscus carpenteri (Dinophyceae) in a tropical marine ecosystem: The first record from the Philippines.

Morphological and phylogenetic analysis showed that the Gambierdiscus isolate from Bolinao, Philippines belongs to the species of G. carpenteri. It was morphologically more similar to the Merimbula strain than the subtropical Florida Keys strain. Growth and toxin production were also investigated at varying levels of temperature, salinity, and irradiance. Gambierdiscus are known to grow favorably in a low light environment. However, this study showed high growth rates of G. carpenteri even at high irradiance levels. Generally, cells produced more toxins at lower treatment levels. Highest cellular toxin content recorded was 7.48 ±â€¯0.49 pg Pbtx eq/cell at culture conditions of 25 °C, 100 µmol photons m-2 s-1, and salinity of 26. Growth rate and toxin production data suggest that cells produced more toxins during the slowest growth at certain range of treatments. This information gives insight into how changes in environmental conditions may affect toxin production and growth of G. carpenteri.

Insights into the dynamics of harmful algal blooms in a tropical estuary through an integrated hydrodynamic-Pyrodinium-shellfish model.

In contrast to temperate Harmful Algal Blooms (HABs), knowledge on the mechanisms driving tropical HABs are less well studied. The interaction of a seasonal temperature window, cysts (for certain species) and large-scale transport are some of the key processes in temperate HABs. In the Philippines, HABs occur not along long open coastlines, but in embayments that are highly influenced by run-off and stratification. These embayments are typically also the sites of cultured or wild harvest shellfish and other aquaculture activities. Sorsogon Bay in the northeastern Philippines has experienced prolonged shellfish-harvesting bans due to blooms by Pyrodinium bahamense var. compressum severely affecting the fisheries industry in this area, as well as leading to Paralytic Shellfish Poisoning illnesses and fatalities. A novel integrated model was developed that mechanistically captures the interactions between hydrodynamic conditions, nutrients, the life history (cells and cysts) of Pyrodinium, as well as the cultured shellfish within the bay and their ensuing toxicities due to ingestion of toxic Pyrodinium cells and cysts. This is the second model developed for HABs in the Philippines, and the first to integrate different components of Pyrodinium bloom dynamics. The model is modularly composed of a watershed nutrient and diffusion model, a 3D hydrodynamic model, a Pyrodinium population model and a shellfish toxin model. It was able to capture the observed temporal variations of Pyrodinium and shellfish toxicity. It was also able to represent some aspects of the spatial distribution in Sorsogon Bay though there were discrepancies. To explore the dynamics of blooms, the linkages between the bloom and decline of the Pyrodinium population with shellfish toxicity as affected by temperature, salinity and nutrients were investigated. Comparisons with field results showed the seasonality of blooms in Sorsogon Bay is driven by increased rainfall. The timing of these conditions is important in facilitating Pyrodinium excystment and reproduction. Model results showed as well the potential significance of shellfish grazing and dinoflagellate cell mortality in influencing the decline of the bloom, and toxicity levels. This approach is promising in helping to understand mechanisms for HABs more holistically, and the model can be further improved to provide more precise quantitative information.