Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam.

Ciguatera fish poisoning (CFP) is a type of food poisoning caused by the consumption of a variety of toxic ciguatera fish species in the tropical and subtropical waters. Although there have been a large number of suspected CFP cases in the Southeast Asian countries, few were confirmed with causative ciguatoxins (CTXs), and reliable information on the symptoms still remains rather limited. In the present study, CTXs in red snapper Lutjanus bohar, implicated in two suspected CFP cases in Vietnam in 2014 and 2016, were determined by use of the single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Ciguatoxin-1B (CTX-1B), 54-deoxyCTX-1B, and 52-epi-54-deoxyCTX-1B were detected in the red snapper by our LC/MS method. Moreover, CTX-1B, 54-deoxyCTX-1B, and 52-epi-54-deoxyCTX-1B were further identified by the time of flight (TOF) LC/MS with the exact mass spectrum. The CTX profile of the red snapper in Vietnam is similar to those of ciguatera fish from Australia, Okinawa Islands in Japan, Kiribati, and Hong Kong. This is the first comprehensive report unambiguously identifying the causative toxins in fish implicated with reliable information on the poisoning symptoms in CFP in Vietnam and/or Southeast Asian countries.

Dactylodinium pterobelotum gen. et sp. nov., a new marine woloszynskioid dinoflagellate positioned between the two families Borghiellaceae and Suessiaceae.

A new marine woloszynskioid dinoflagellate Dactylodinium pterobelotum gen. et sp. nov., collected from a southern Vietnamese estuary, was described on the basis of LM, SEM, and TEM, and molecular phylogeny inferred from rDNA sequences. This species had the smallest number of amphiesmal vesicles (5 latitudinal series) in woloszynskioid dinoflagellates assigned to the Suessiaceae and Borghiellaceae. The eyespot was of type B, composed of osmiophilic globules and brick-like material, located in- and outside of the chloroplast respectively. An apical structure comprised a pair of elongate anterior vesicles (PEV). A large peduncle was conspicuous, located in the sulcal extension in the epicone, and supported by a microtubular strand of ~140 microtubules. Ultrastructural features of trichocysts represent a novel type in the Dinophyceae, bearing lateral hairs besides anterior fibers. The molecular phylogeny based on partial LSU rDNA showed the species in a basal position in the family Suessiaceae; this indicates the eyespot type B and PEV of the Borghiellaceae are ancestral states of the eyespot comprising brick-like material (type E) and an elongate apical vesicle of the Suessiaceae.

Changes in microbial communities, including both uncultured and culturable bacteria, with mid-ocean ballast-water exchange during a voyage from Japan to Australia.

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.

SPECIES BOUNDARIES IN THE TOXIC DINOFLAGELLATE PROROCENTRUM LIMA (DINOPHYCEAE, PROROCENTRALES), BASED ON MORPHOLOGICAL AND PHYLOGENETIC CHARACTERS(1).

Wild and cultured specimens of Prorocentrum lima (Ehrenb.) F. Stein from 26 widely different areas in 13 countries were examined in order to determine consistent characters for delimiting species boundaries in this taxon. The morphological characters valve shape, valve size, valve ornamentation, number and shape of valve pores, number and shape of marginal pores, and periflagellar platelets were observed using LM and SEM, and two molecular genetic regions were sequenced. We identified stable morphological characters that were consistent among wild specimens and all cultures, which were valve shape, valve ornamentation, and number and arrangement of periflagellar platelets. All cultures of P. lima identified by these characters formed a monophyletic group in phylogenetic analyses based on the two genes, which, however, included the species Prorocentrum arenarium. P. arenarium was determined to be within the range of morphological variation of P. lima, and therefore we synonymize the two taxa. Within this monophyletic group, P. lima was divided into several subclades in the all phylogenetic analyses. There were no morphological characters specifically related to any one subclade. The subclades appeared to correlate broadly to sample collection regions, suggesting that geographically separated populations may have become genetically distinct within this epi-benthic species. We have emended species boundaries in P. lima.

Denaturing gradient gel electrophoresis shows that bacterial communities change with mid-ocean ballast water exchange.

Ships carry ballast water for better stability and to control trim. However, the discharge of ballast water near ports is known to transport invasive species from one coastal area to another. The exchange of ballast water on the high seas is supposed to reduce such invasions of exotic species. In this study, we used denaturing gradient gel electrophoresis (DGGE) to analyze the composition of the bacterial community in ballast water before and after such a mid-ocean exchange, and we also measured total bacterial counts. Our findings confirmed that the ballast water was replaced by the mid-ocean exchange, as indicated by the marked change in the composition of the bacterial community. There was also a significant decrease in bacterial abundance after the mid-ocean exchange. Finally, our findings support the incubation hypothesis, because the composition of the bacterial communities changed over time within the same ballast water.

Are prorocentroid dinoflagellates monophyletic? A study of 25 species based on nuclear and mitochondrial genes.

The dinoflagellate sub-class Prorocentrophycidae has a distinct morphology, lacking the typical dinoflagellate cell structure of a clear cingulum and sulcus. It includes species that produce the toxin okadaic acid. Despite its uniqueness, the group has been found polyphyletic in some previous molecular phylogenetic studies. We have re-investigated the phylogeny of this sub-class by culturing and sequencing new strains, comparing sequences from three genes, the mitochondrial cytochrome c oxidase subunit 1 (cox 1) and the nuclear large and small subunit rRNA (LSU and SSU) encoding genes. We analyzed sequences from twenty-five named and two still undescribed species of Prorocentrophycidae. We used newly recognized features of the secondary structure to align regions of the LSU rRNA. The phylogeny based on cox 1 provided the most well-supported tree and showed strong support for the monophyly of prorocentroid dinoflagellates, while the LSU phylogeny was inconclusive. As in previous studies, phylogeny based on SSU shows the group to appear paraphyletic, however, support values were low. Two strongly supported sub-clades were consistently identified. Benthic and planktonic modes appear to have evolved on multiple occasions within both clades of Prorocentriphycidae. The capability to synthesize toxins appears to have arisen early in prorocentroid evolution and, in particular, okadaic acid synthesis is present in some, but not all, members of Clade 2. The D2a region of the LSU rRNA appears to have developed a deletion in three definable steps during prorocentroid evolution. While the phylogenies inferred from the three genes were not congruent, our results give reserved support to the monophyly of the group.

Ocean urea fertilization for carbon credits poses high ecological risks.

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.

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.

Production of isodomoic acids A and B as major toxin components of a pennate diatom Nitzschia navis-varingica.

In order to obtain more detailed information on the distribution of domoic acid-producing Nitzschia in Asian waters, Nitzschia-like diatoms were isolated primarily from the Philippines and established in culture for analysis by HPLC with fluorescence detection. Out of 58 isolates, 35 Nitzschia-like diatoms from the estuary areas of San Pedro Bay, Tacloban City and Manila Bay showed significant levels of domoic acid production (1.3-5.6pg/cell). These were identified as Nitzschia navis-varingica. Two isolates from the same locality did not produce domoic acid. Of the 21 isolates from Bulacan Estuary, Manila Bay, none produced domoic acid. They did, however, produce two substances that seemed to be domoic acid derivatives. One of the strains was mass cultured and the substances were extracted, purified and analyzed by LC-MS/MS, proton and (13)C NMR, and UV spectra. The produced substances were determined as isodomoic acids A and B. This is the first report of a diatom that produces isodomoic acids A and B as major toxin components.