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Molecular-based approaches for harmful algal bloom species, which can be difficult to identify by light microscopy, are important tools for detecting and predicting the blooms. In this study, 84 samples from Aotearoa/New Zealand coastal waters that contained Azadinium-like cells were analysed using an Amphidomataceae real-time PCR assay. Nineteen samples were positive, and 24 Azadinium-like clonal isolates were established from these samples. Subsequently, only five isolates from one sampling point were positive using the real-time PCR assay. Four of the five isolates were genetically identified as Amphidoma languida and one as Azadinium dalianense. The field samples were analysed retrospectively using the species-specific real-time PCR assays; Am. languida was detected from the temperate areas, while Az. poporum and Az. spinosum were detected at one site. Two representative strains of each species were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including Am. languida, which is known as toxic in the previous studies, but neither produced the currently monitored azaspiracids (AZAs). These two newly recorded species for New Zealand are important findings. The results highlight the need for more information on the spatio-temporal variations of potentially toxic Amphidomataceae species and the risk of AZAs in New Zealand coastal waters. [ABSTRACT FROM AUTHOR]

Planktonic dinoflagellate records for New Zealand are substantial due to intense monitoring programmes that have taken place on behalf of New Zealand's biotoxin regulators and the shellfish industry since 1993. At that time a Karenia bloom caused human illnesses and shellfish harvesting ceased until monitoring was instigated. Phytoplankton records are based on morphological identification using light microscopy but are backed up by government funded research programmes which implement techniques such as electron microscopy, DNA sequencing, molecular detection assays and high throughput sequencing of environmental DNA. This checklist will support management of aquaculture industries, recreational shellfish harvests and environmental health initiatives. Some genera are considered benthic or epiphytic, but have lengthy planktonic life stages. Forty-five genera in the class Dinophyceae Fritsch are reported: Akashiwo, Alexandrium, Amphidinium, Amylax, Azadinium, Biecheleria, Bysmatrum, Cachonina, Cochlodinium (synonym: Margalefidinium), Coolia, Dicroerisma, Dinophysis, Diploneis, Diplopsalis, Fragilidium, Glenodinium, Gonyaulax, Gymnodinium, Gyrodinium, Heterocapsa, Karenia, Karlodinium, Lepidodinium, Lingulodinium, Margalefidinium, Ostreopsis, Oxyphysis, Pelagodinium, Pentapharsodinium, Phalacroma, Podolampas, Polarella, Polykrikos, Prorocentrum, Protodinium, Protoodinium, Protoperidinium, Pseliodinium, Scrippsiella, Takayama, Togula, Torodinium, Tripos, Vulcanodinium, Wolosynska. Other genera belonging to the Infraphylum Dinoflagellata also occur in New Zealand waters. They are not in the list but include Noctiluca, Pronoctiluca and Spatulodinium (Class: Noctilucophyceae) and Oxyrrhis (Class: Oxyrrhidophyceae). [ABSTRACT FROM AUTHOR]

Ciguatera Fish Poisoning (CFP) is increasing across the Pacific and the distribution of the causative dinoflagellates appears to be expanding. Subtle differences in thecal plate morphology are used to distinguish dinoflagellate species, which are difficult to determine using light microscopy. For these reasons we sought to develop a Quantitative PCR assay that would detect all species from both Gambierdiscus and Fukuyoa genera in order to rapidly screen environmental samples for potentially toxic species. Additionally, a specific assay for F. paulensis was developed as this species is of concern in New Zealand coastal waters. Using the assays we analyzed 31 samples from three locations around New Zealand and the Kingdom of Tonga. Fourteen samples in total were positive for Gambierdiscus/Fukuyoa and two samples were also positive using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using high-throughput sequencing metabarcoding. The majority of reads corresponded to Gambierdiscus species with three species identified at all sites (G. australes, G. honu and G. polynesiensis). This is the first confirmed identification of G. polynesiensis, a known ciguatoxin producer, in New Zealand waters. Other known toxin-producing genera were also detected, included Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis, and Prorocentrum. [ABSTRACT FROM AUTHOR]

Species in the genus Gambierdiscus produce ciguatoxins (CTXs) and/or maitotoxins (MTXs), which may cause ciguatera fish poisoning (CFP) in humans if contaminated fish are consumed. Species of Gambierdiscus have previously been isolated from macroalgae at Rangitahua (Raoul Island and North Meyer Islands, northern Kermadec Islands), and the opportunity was taken to sample for Gambierdiscus at the more southerly Macauley Island during an expedition in 2016. Gambierdiscus cells were isolated, cultured, and DNA extracted and sequenced to determine the species present. Bulk cultures were tested for CTXs and MTXs by liquid chromatography-mass spectrometry (LC-MS/MS). The species isolated were G. australes, which produced MTX-1 (ranging from 3 to 36 pg/cell), and G. polynesiensis, which produced neither MTX-1 nor, unusually, any known CTXs. Isolates of both species produced putative MTX-3. The risk of fish, particularly herbivorous fish, causing CFP in the Zealandia and Kermadec Islands region is real, although in mainland New Zealand the risk is currently low. Both Gambierdiscus and Fukuyoa have been recorded in the sub-tropical northern region of New Zealand, and so the risk may increase with warming seas and shift in the distribution of Gambierdiscus species. [ABSTRACT FROM AUTHOR]

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G. honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration. [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning results from consumption of seafood naturally contaminated by saxitoxin and its congeners, the paralytic shellfish toxins (PSTs). The levels of such toxins are regulated internationally, and maximum permitted concentrations in seafood have been established in many countries. A mouse bioassay is an approved method for estimating the levels of PSTs in seafood, but this is now being superseded in many countries by instrumental methods of analysis. Such analyses provide data on the levels of many PSTs in seafood, but for risk assessment, knowledge of the relative toxicities of the congeners is required. These are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are largely based on relative specific activities following intraperitoneal injection in a mouse bioassay rather than on acute toxicity determinations. A more relevant parameter for comparison would be median lethal doses via oral administration, since this is the route through which humans are exposed to PSTs. In the present study, the median lethal doses of gonyautoxin 5, gonyautoxin 6, decarbamoyl neosaxitoxin and of equilibrium mixtures of decarbamoyl gonyautoxins 2&3, C1&2 and C3&4 by oral administration to mice have been determined and compared with toxicities via intraperitoneal injection. The results indicate that the TEFs of several of these substances require revision in order to more accurately reflect the risk these toxins present to human health. [ABSTRACT FROM AUTHOR]

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples. [ABSTRACT FROM AUTHOR]