Geographical distribution, molecular and toxin diversity of the dinoflagellate species Gambierdiscus honu in the Pacific region.

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns.

Unraveling the Gonyaulax baltica Species Complex: Cyst-theca Relationship of Impagidinium variaseptum, Spiniferites pseudodelicatus sp. nov. and S. ristingensis (Gonyaulacaceae, Dinophyceae), With Descriptions of Gonyaulax bohaiensis sp. nov, G. amoyensis sp. nov. and G. portimonensis sp. nov.

The taxonomy of the extant dinoflagellate genus Gonyaulax is challenging since its thecate morphology is rather conservative. In contrast, cysts of Gonyaulax are varied in morphology and have been related with the fossil-based genera Spiniferites and Impagidinium. To better understand the systematics of Gonyaulax species, we performed germination experiments on cysts that can be identified as S. ristingensis, an unidentified Spiniferites with petaloid processes here described as Spiniferites pseudodelicatus sp. nov. and Impagidinium variaseptum from Chinese and Portuguese waters. Despite marked differences in cyst morphology, motile cells of S. pseudodelicatus and I. variaseptum are indistinguishable from Gonyaulax baltica. Motile cells hatched from S. ristingensis are morphologically similar to G. baltica as well but differ in the presence of one pronounced antapical spine. Three new species, Gonyaulax amoyensis (cyst equivalent S. pseudodelicatus), Gonyaulax bohaiensis (cyst equivalent I. variaseptum), and Gonyaulax portimonensis (cyst equivalent S. ristingensis), were erected. In addition, a new ribotype (B) of G. baltica was reported from South Korea and a bloom of G. baltica ribotype B is reported from New Zealand. Molecular phylogeny based on LSU and SSU rRNA gene sequences revealed that Gonyaulax species with minute or short antapical spines formed a well-resolved clade, whereas species with two pronounced antapical spines or lack of antapical spines formed the sister clade. Six strains of four above species were examined for yessotoxin production by liquid chromatography coupled with tandem mass spectrometry, and very low concentrations of yessotoxin were detected for one G. bohaiensis strain.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Assessment of Ciguatera and Other Phycotoxin-Related Risks in Anaho Bay (Nuku Hiva Island, French Polynesia): Molecular, Toxicological, and Chemical Analyses of Passive Samplers.

Ciguatera poisoning is a foodborne illness caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates from the genera Gambierdiscus and Fukuyoa. The suitability of Solid Phase Adsorption Toxin Tracking (SPATT) technology for the monitoring of dissolved CTXs in the marine environment has recently been demonstrated. To refine the use of this passive monitoring tool in ciguateric areas, the effects of deployment time and sampler format on the adsorption of CTXs by HP20 resin were assessed in Anaho Bay (Nuku Hiva Island, French Polynesia), a well-known ciguatera hotspot. Toxicity data assessed by means of the mouse neuroblastoma cell-based assay (CBA-N2a) showed that a 24 h deployment of 2.5 g of resin allowed concentrating quantifiable amounts of CTXs on SPATT samplers. The CTX levels varied with increasing deployment time, resin load, and surface area. In addition to CTXs, okadaic acid (OA) and dinophysistoxin-1 (DTX1) were also detected in SPATT extracts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), consistent with the presence of Gambierdiscus and Prorocentrum species in the environment, as assessed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTS) metabarcoding analyses conducted on passive window screen (WS) artificial substrate samples. Although these preliminary findings await further confirmation in follow-up studies, they highlight the usefulness of SPATT samplers in the routine surveillance of CP risk on a temporal scale, and the monitoring of other phycotoxin-related risks in ciguatera-prone areas.

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data.

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20-25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

qPCR Assays for the Detection and Quantification of Multiple Paralytic Shellfish Toxin-Producing Species of Alexandrium.

Paralytic shellfish toxin producing dinoflagellates have negatively impacted the shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former Alexandrium tamarense species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for A. australiense, and new primers targeting A. ostenfeldii, A. catenella, and A. pacificum. We showed that our new primers for A. pacificum are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the A. catenella and A. australiense assays, 2 cells/L and 1 cyst/mg sediment for the A. pacificum assay, and 1 cells/L for the A. ostenfeldii assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring A. catenella, A. pacificum, and A. australiense in samples from the east coast of Tasmania, Australia.