Taxonomy and toxicity of a bloom-forming Ostreopsis species (Dinophyceae, Gonyaulacales) in Tahiti island (South Pacific Ocean): one step further towards resolving the identity of O. siamensis.

Among dinoflagellates responsible for benthic harmful algal blooms, the genus Ostreopsis primarily described from tropical areas has been increasingly reported from subtropical and temperate areas worldwide. Several species of this toxigenic genus produce analogs of palytoxin, thus representing a major threat to human and environmental health. The taxonomy of several species needs to be clarified as it was based mostly on morphological descriptions leading in some cases to ambiguous interpretations and misidentifications. The present study aims at reporting a benthic bloom that occurred in April 2019 in Tahiti island, French Polynesia. A complete taxonomic investigation of the blooming Ostreopsis species was realized using light, epifluorescence and field emission electron microscopy and phylogenetic analyses inferred from LSU rDNA and ITS-5.8S rDNA regions. Toxicity of a natural sample and strains isolated from the bloom was assessed using both neuroblastoma cell-based assay and LC-MS/MS analyses. Morphological observations showed that cells were round to oval, large, 58.0-82.5 µm deep (dorso-ventral length) and 45.7-61.2 µm wide. The cingulum was conspicuously undulated, forming a 'V' in ventral view. Thecal plates possessed large pores in depressions, with a collar rim. Detailed observation also revealed the presence of small thecal pores invisible in LM. Phylogenetic analyses were congruent and all sequences clustered within the genotype Ostreopsis sp. 6, in a subclade closely related to sequences from the Gulf of Thailand and Malaysia. No toxicity was found on the field sample but all the strains isolated from the bloom were found to be cytotoxic and produced ostreocin D, a lower amount of ostreocins A and B and putatively other compounds. Phylogenetic data demonstrate the presence of this species in the Gulf of Thailand, at the type locality of O. siamensis, and morphological data are congruent with the original description and support this identification.

Unsuspected intraspecific variability in the toxin production, growth and morphology of the dinoflagellate Alexandrium pacificum R.W. Litaker (Group IV) blooming in a South Western Mediterranean marine ecosystem, Annaba Bay (Algeria).

Physiological plasticity gives HABs species the ability to respond to variations in the surrounding environment. The aim of this study was to examine morphological and physiological variability in Alexandrium pacificum R.W. Litaker (Group IV) (former Alexandrium catenella) blooming in Annaba bay, Algeria. Monoclonal cultures of up to 30 strains of this neurotoxic dinoflagellate were established by the germination of single resting cysts from the surface sediment of this southern Mediterranean marine ecosystem. Ribotyping confirmed formally for the first time that A. pacificum is developing in Eastern Algerian waters. Toxin analyses of A. pacificum strains revealed substantial intraspecific variability in both the profile and toxin amount. However, the toxin profile of most strains is characterized by the dominance of GTX6 (up to 96 mol %) which is the less toxic paralytic molecule. The toxin concentrations in the isolated strains varied widely between 3.8 and 30.82 fmol cell-1. We observed an important variation in the growth rate of the studied A. pacificum strains with values ranging from 0.05 to 0.33 d-1. The lag time of the studied strains varied widely and ranged from 4 to 20 days. The intraspecific diversity could be a response to the selection pressure which may be exerted by different environmental conditions over time and which can be genetically and in turn physiologically expressed. This study highlights, for the first time, that the sediment of a limited area holds an important diversity of A. pacificum cysts which give when germinate populations with noticeable physiological plasticity. Consequently, this diversified natural populations allow an exceptional adaptation to specific environmental conditions to outcompete local microalgae and to establish HABs which could explain why this dinoflagellate is successful and expanding worldwide.

Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity.

To date, the genus Ostreopsis comprises eleven described species, of which seven are toxigenic and produce various compounds presenting a major threat to human and environmental health. The taxonomy of several of these species however remains controversial, as it was based mostly on morphological descriptions leading, in some cases, to ambiguous interpretations and even possible misidentifications. The species Ostreopsis lenticularis was first described by Y. Fukuyo from French Polynesia using light microscopy observations, but without genetic information associated. The present study aims at revisiting the morphology, molecular phylogeny and toxicity of O. lenticularis based on the analysis of 47 strains isolated from 4 distinct locales of French Polynesia, namely the Society, Australes, Marquesas and Gambier archipelagos. Observations in light, epifluorescence and field emission scanning electron microscopy of several of these strains analyzed revealed morphological features in perfect agreement with the original description of O. lenticularis. Cells were oval, not undulated, 60.5-94.4 µm in dorso-ventral length, 56.1-78.2 µm in width, and possessed a typical plate pattern with thecal plates showing two sizes of pores. Phylogenetic analyses inferred from the LSU rDNA and ITS-5.8S sequences revealed that the 47 strains correspond to a single genotype, clustering with a strong support with sequences previously ascribed to Ostreopsis sp. 5. Clonal cultures of O. lenticularis were also established and further tested for their toxicity using the neuroblastoma cell-based assay and LCMS/MS analyses. None of the 19 strains tested showed toxic activity on neuroblastoma cells, while LCMS/MS analyses performed on the strains from Tahiti Island (i.e. type locality) confirmed that palytoxin and related structural analogs were below the detection limit. These findings allow to clarify unambiguously the genetic identity of O. lenticularis while confirming previous results from the Western Pacific which indicate that this species shows no toxicity, thus stressing the need to reconsider its current classification within the group of toxic species.

Tissue Distribution and Elimination of Ciguatoxins in Tridacna maxima (Tridacnidae, Bivalvia) Fed Gambierdiscus polynesiensis.

Ciguatera is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Ciguatera-like poisoning events involving giant clams (Tridacna maxima) are reported occasionally from Pacific islands communities. The present study aimed at providing insights into CTXs tissue distribution and detoxification rate in giant clams exposed to toxic cells of Gambierdiscus polynesiensis, in the framework of seafood safety assessment. In a first experiment, three groups of tissue (viscera, flesh and mantle) were dissected from exposed individuals, and analyzed for their toxicity using the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The viscera, flesh, and mantle were shown to retain 65%, 25%, and 10% of the total toxin burden, respectively. All tissues reached levels above the safety limit recommended for human consumption, suggesting that evisceration alone, a practice widely used among local populations, is not enough to ensure seafood safety. In a second experiment, the toxin content in contaminated giant clams was followed at different time points (0, 2, 4, and 6 days post-exposure). Observations suggest that no toxin elimination is visible in T. maxima throughout 6 days of detoxification.

Toxicological Investigations on the Sea Urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the Presence of Pacific Ciguatoxins.

The sea urchin Tripneustes gratilla (Toxopneustidae, Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T.gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T.gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G.polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T.gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T.gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology.

Effects of copper and butyltin compounds on the growth, photosynthetic activity and toxin production of two HAB dinoflagellates: The planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata.

Controlled laboratory experiments were conducted to test the effects of copper (Cu2+) and butyltins (BuT) on the growth, photosynthetic activity and toxin content of two HABs (Harmful Algal Blooms) dinoflagellates, the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata. Microalgae were exposed to increasing concentrations of Cu2+ (10-4 to 31 nM) or BuT (0.084 to 84 nM) for seven days. When considering the growth, EC50 values were 0.16 (±0.09) nM and 0.03 (±0.02) nM of Cu2+ for A. catenella and O. cf. ovata, respectively. Regarding BuT, EC50 was 14.2 (±6) nM for O. cf. ovata, while A. catenella growth inhibition appeared at BuT concentrations ≥27 nM. Photosynthetic activity of the studied dinoflagellates decreased with increasing Cu and BuT concentrations. For O. cf. ovata, the response of this physiological parameter to contamination was less sensitive than the biomass. Cu exposure induced the formation of temporary cysts in both organisms that could resist adverse conditions. The ovatoxin-a and -b concentrations in O. cf. ovata cells increased significantly in the presence of Cu. Altogether, the results suggest a better tolerance of the planktonic A. catenella to Cu and BuT. This could result in a differentiated selection pressure exerted by these metals on phytoplankton species in highly polluted waters. The over-production of toxins in response to Cu stress could pose supplementary health and socio-economic threats in the contaminated marine ecosystems where HABs develop.

Tectus niloticus (Tegulidae, Gastropod) as a Novel Vector of Ciguatera Poisoning: Detection of Pacific Ciguatoxins in Toxic Samples from Nuku Hiva Island (French Polynesia).

Ciguatera fish poisoning (CFP) is a foodborne disease caused by the consumption of seafood (fish and marine invertebrates) contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genus Gambierdiscus. The report of a CFP-like mass-poisoning outbreak following the consumption of Tectus niloticus (Tegulidae, Gastropod) from Anaho Bay on Nuku Hiva Island (Marquesas archipelago, French Polynesia) prompted field investigations to assess the presence of CTXs in T. niloticus. Samples were collected from Anaho Bay, 1, 6 and 28 months after this poisoning outbreak, as well as in Taiohae and Taipivai bays. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of CTXs only in Anaho Bay T. niloticus samples. This is consistent with qPCR results on window screen samples indicating the presence of Gambierdiscus communities dominated by the species G. polynesiensis in Anaho Bay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major congener, followed by P-CTX-3C, P-CTX-4A and P-CTX-4B in toxic samples. Between July 2014 and November 2016, toxin content in T. niloticus progressively decreased, but was consistently above the safety limit recommended for human consumption. This study confirms for the first time T. niloticus as a novel vector of CFP in French Polynesia.

Evidence of the bioaccumulation of ciguatoxins in giant clams (Tridacna maxima) exposed to Gambierdiscus spp. cells.

Ciguatera Fish Poisoning (CFP) is a foodborne disease classically related to the consumption of tropical coral reef fishes contaminated with ciguatoxins (CTXs), neurotoxins produced by dinoflagellates of the Gambierdiscus genus. Severe atypical ciguatera-like incidents involving giant clams, a marine resource highly consumed in the South Pacific, are also frequently reported in many Pacific Islands Countries and Territories. The present study was designed to assess the ability of giant clams to accumulate CTXs in their tissues and highlight the potential health risks associated with their consumption. Since giant clams are likely to be exposed to both free-swimming Gambierdiscus cells and dissolved CTXs in natural environment, ex situ contamination experiments were conducted as follows: giant clams were exposed to live or lyzed cells of TB92, a highly toxic strain of G. polynesiensis containing 5.83±0.85pg P-CTX-3C equiv.cell-1vs. HIT0, a weakly toxic strain of G. toxicus containing only (2.05±1.16)×10-3pg P-CTX-3C equiv.cell-1, administered over a 48h period at a concentration of 150cellsmL-1. The presence of CTXs in giant clams tissues was further assessed using the mouse neuroblastoma cell-based assay (CBA-N2a). Results showed that giant clams exposed to either lyzed or live cells of TB92 were able to bioaccumulate CTXs at concentrations well above the safety limit recommended for human consumption, i.e. 3.28±1.37 and 2.92±1.03ng P-CTX-3C equiv.g-1 flesh (wet weight), respectively, which represented approximately 3% of the total toxin load administered to the animals. In contrast, giant clams exposed to live or lyzed cells of HIT0 were found to be free of toxins, suggesting that in the nature, the risk of contamination of these bivalves is established only in the presence of highly toxic blooms of Gambierdiscus. Liquid chromatography-mass spectrometry (LC-MS/MS) analyses confirmed CBA-N2a results and also revealed that P-CTX-3B was the major CTX congener retained in the tissues of giant clams fed with TB92 cells. To the best of our knowledge, this study is the first to provide evidence of the bioaccumulation of Gambierdiscus CTXs in giant clams and confirms that these bivalve molluscs can actually constitute another pathway in ciguatera poisonings. While most monitoring programs currently focus on fish toxicity, these findings stress the importance of a concomitant surveillance of these marine invertebrates in applicable locations for an accurate assessment of ciguatera risk.

A feedback mechanism to control apoptosis occurs in the digestive gland of the oyster crassostrea gigas exposed to the paralytic shellfish toxins producer Alexandrium catenella.

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs.

First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium.

Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in the New Caledonian lagoon. The possible role played by Trichodesmium blooms in the development of clupeotoxism, this human intoxication following the ingestion of plankton-eating fish and classically associated with Ostreopsis blooms, is also discussed.