Effects of Harmful Algal Blooms on Fish and Shellfish Species: A Case Study of New Zealand in a Changing Environment.

Harmful algal blooms (HABs) have wide-ranging environmental impacts, including on aquatic species of social and commercial importance. In New Zealand (NZ), strategic growth of the aquaculture industry could be adversely affected by the occurrence of HABs. This review examines HAB species which are known to bloom both globally and in NZ and their effects on commercially important shellfish and fish species. Blooms of Karenia spp. have frequently been associated with mortalities of both fish and shellfish in NZ and the sub-lethal effects of other genera, notably Alexandrium spp., on shellfish (which includes paralysis, a lack of byssus production, and reduced growth) are also of concern. Climate change and anthropogenic impacts may alter HAB population structure and dynamics, as well as the physiological responses of fish and shellfish, potentially further compromising aquatic species. Those HAB species which have been detected in NZ and have the potential to bloom and harm marine life in the future are also discussed. The use of environmental DNA (eDNA) and relevant bioassays are practical tools which enable early detection of novel, problem HAB species and rapid toxin/HAB screening, and new data from HAB monitoring of aquaculture production sites using eDNA are presented. As aquaculture grows to supply a sizable proportion of the world's protein, the effects of HABs in reducing productivity is of increasing significance. Research into the multiple stressor effects of climate change and HABs on cultured species and using local, recent, HAB strains is needed to accurately assess effects and inform stock management strategies.

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.

Overview of Australian and New Zealand harmful algal species occurrences and their societal impacts in the period 1985 to 2018, including a compilation of historic records.

Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

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.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates.

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

A Long-Term Time Series of Dinophysis acuminata Blooms and Associated Shellfish Toxin Contamination in Port Underwood, Marlborough Sounds, New Zealand.

Blooms of the dinoflagellate Dinophysis acuminata occur every year in an important mussel cultivation area in Port Underwood, Marlborough Sounds, New Zealand. Annual maximum cell numbers range from 1500⁻75,000 cells L-1 and over 25 years of weekly monitoring the D. acuminata bloom has never failed to exhibit peaks in abundance at some time between spring and autumn. During winter (June⁻August) the dinoflagellate is often undetectable, or at low levels (≤100 cells L-1), and the risk of diarrhetic shellfish poisoning (DSP)-toxin contamination over this period is negligible. Bloom occurrence may be coupled to the abundance of D. acuminata prey (Mesodinium sp.) but the mechanism by which it maintains its long-term residence in this hydrologically dynamic environment is unknown. The toxin profile of D. acuminata is dominated by pectenotoxin-2 (PTX-2) and dinophysistoxin-1 (DTX-1), but the cellular toxin content is low. It is rare that free DTX-1 is detected in mussels as this is invariably exclusively present as fatty acid-esters. In only five out of >2500 mussel samples over 16 years have the levels of total DTX-1 marginally exceeded the regulated level of 0.16 mg kg-1. It is also rare that free PTX-2 is detected in mussels, as it is generally only present in its hydrolysed non-toxic PTX-2 seco acid form. The D. acuminata alert level of 1000 cells L-1 is often exceeded without DTX-1 residues increasing appreciably, and this level is considered too conservative.

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.

The use of a mucus trap by Dinophysis acuta for the capture of Mesodinium rubrum prey under culture conditions.

A capture mechanism observed in a culture of the dinoflagellate Dinophysis acuta when preying on the ciliate Mesodinium rubrum (also sometimes referred to as Myrionecta rubra) is described. The dinoflagellate released cohesive clumps of mucilage into the culture media. When M. rubrum cells came into contact with this mucilage, they were immediately immobilized, but remained alive for a short period of time. Observations of D. acuta cells 'visiting and probing' trapped M. rubrum cells were made and at a critical point D. acuta cells removed individual M. rubrum cells from the mucus to swim away with them. The removal of M. rubrum from the mucus coincided with the cells losing all their cilia and becoming swollen, presumably signifying the death of the cell. These changes may enable the D. acuta peduncle to penetrate the ciliate cell cortex. It is hypothesized that toxins produced by D. acuta play a role in the immobilization process within the mucilage trap.

Isolation and characterization of an enzyme from the Greenshell™ mussel Perna canaliculus that hydrolyses pectenotoxins and esters of okadaic acid.

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins.

Development of solid phase adsorption toxin tracking (SPATT) for monitoring anatoxin-a and homoanatoxin-a in river water.

Sampling and monitoring for cyanotoxins can be problematic as concentrations change with environmental and hydrological conditions. Current sampling practices (e.g. grab samples) provide data on cyanotoxins present only at one point in time and may miss areas or times of highest risk. Recent research has identified the widespread distribution of anatoxin-producing benthic cyanobacteria in rivers highlighting the need for development of effective sampling techniques. In this study we evaluated the potential of an in situ method known as solid phase adsorption toxin tracking (SPATT) for collecting and concentrating anatoxin-a (ATX) and homoanatoxin-a (HTX) in river water. Fifteen different adsorption substrates were screened for efficiency of ATX uptake, nine of which retained high proportions (>70%) of ATX. Four substrates were then selected for a 24-h trial in a SPATT bag format in the laboratory. The greatest decrease in ATX in the water was observed with powdered activated carbon (PAC) and Strata-X (a polymeric resin) SPATT bags. A 3-d field study in a river containing toxic benthic cyanobacterial mats was undertaken using PAC and Strata-X SPATT bags. ATX and HTX were detected in all SPATT bags. Surface grab samples were taken throughout the field study and ATX and HTX were only detected in one of the water samples, highlighting the limitations of this currently used method. Both Strata-X and PAC were found to be effective absorbent substrates. PAC has the advantage that it is cheap and readily available and appears to continue to sorb toxins over longer periods than Strata-X. SPATT has the potential to be integrated into current cyanobacterial monitoring programmes and would be a very useful and economical tool for early warning of ATX and HTX contamination in water.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand.

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.

In situ passive solid-phase adsorption of micro-algal biotoxins as a monitoring tool.

Laboratory and field studies of the passive solid-phase adsorption toxin tracking (SPATT) method have been carried out around the world. A wide range of marine micro-algal toxins have been detected and the potential of the method to provide reliable, sensitive, time-integrated sampling to monitor the occurrence of toxic algal bloom events has been demonstrated. The method has several important advantages over current phytoplankton and shellfish monitoring methods. Trials of various adsorption substrates have been carried out and the best candidates have been selected for the lipophilic marine biotoxin groups; however, research continues to locate suitable substrates for the more polar water-soluble compounds such as domoic acid and the saxitoxins. The technique has also been successfully applied to the detection of a range of freshwater cyanobacterial toxins.

Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves.

A simple and sensitive in situ method for monitoring the occurrence of toxic algal blooms and shellfish contamination events has been developed. The technique involves the passive adsorption of biotoxins onto porous synthetic resin filled sachets (SPATT bags) and their subsequent extraction and analysis. The success of the method is founded on the observation that during algal blooms significant amounts of toxin, including the low polarity lipophilic compounds such as the pectenotoxins and the okadaic acid complex toxins, are dissolved in the seawater. The results of field trials during Dinophysis acuminata and Protoceratium reticulatum blooms are presented. These data prove the concept and demonstrate that the technique provides a means of forecasting shellfish contamination events and predicting the net accumulation of polyether toxins by mussels. As an early warning method it has many advantages over current monitoring techniques such as shellfish-flesh testing and phytoplankton monitoring. In contrast to the circumstantial evidence provided by genetic probe technologies and conventional phytoplankton monitoring methods, it directly targets the toxic compounds of interest. The extracts that are obtained for analysis lack many of the extraneous lipophilic materials in crude shellfish extracts so that many of the matrix problems associated with chemical and biological analysis of these extracts are eliminated. Analyses can confidently target parent compounds only, because analytical and toxicological uncertainties associated with the multiplicity of toxin analogues produced by in vivo biotransformation in shellfish tissues are reduced. Time integrated sampling provides a good simulation of biotoxin accumulation in filter feeders and the high sensitivity provides lengthy early warning and conservative estimates of contamination potential. The technique may reduce monitoring costs and provide improved spatial and temporal sampling opportunities. When coupled with appropriate analytical techniques (e.g. LC-MS/MS multi-toxin screens, ELISA assays, receptor binding assays), the technique has the potential to offer a universal early warning method for marine and freshwater micro-algae toxins.

Acute toxicity of gymnodimine to mice.

The acute toxicity of the phycotoxin gymnodimine to female Swiss mice by intraperitoneal injection and by oral administration has been determined. Gymnodimine was highly toxic by injection, the LD50 being only 96 microg/kg. Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects. Gymnodimine was also toxic after oral administration by gavage (LD50 755 microg/kg), but was much less toxic when administered with food. No signs of toxicity were seen in mice voluntarily ingesting food containing gymnodimine at a level sufficient to give a dose of approximately 7500 microg/kg. Pre-treatment with physostigmine or neostigmine protected against injected gymnodimine, suggesting that the latter exerts its toxic effects via blockade of nicotinic receptors at the neuromuscular junction. The low toxicity of gymnodimine when ingested with food suggests that this compound is of low risk to humans, a conclusion that is consonant with anecdotal evidence for the absence of harmful effects in individuals consuming shellfish contaminated with gymnodimine.

Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand.

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS-MS). Using a C8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC-MS-MS spectra obtained for the [M+NH4]- ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC-MS-MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC-MS/MS analysis.

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.