Harmful Algae 34 (2014) 36–41

Epiphytic dinoflagellates in sub-tropical New Zealand, in particular thegenus Coolia Meunier

Lesley Rhodes a,*, Kirsty Smith a, Gemma Gimenez Papiol a, Janet Adamson a,Tim Harwood a, Rex Munday b

a Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson 7042, New Zealandb AgResearch, Ruakura Agricultural Research Centre, 10 Bisley Road, Private Bag 3240, Hamilton, New Zealand

A R T I C L E I N F O

Article history:

Received 16 September 2013

Received in revised form 12 February 2014

Accepted 13 February 2014

Keywords:

Coolia monotis

Coolia malayensis

Ostreopsis, palytoxin

Amphidinium

Gambierdiscus

New Zealand

A B S T R A C T

Macroalgae growing in New Zealand’s sub-tropical waters were sampled for epiphytic microalgae, in

particular dinoflagellates. Four new Coolia isolates collected from sites throughout Northland in January/

February 2013 were all identified as C. malayensis as determined by large subunit ribosomal DNA (LSU

rDNA) sequence analysis and scanning electron microscopy. C. malayensis, a common dinoflagellate

species in New Zealand, was previously reported as C. monotis and isolates held in the Cawthron Institute

Culture Collection of Microalgae have now been reclassified based on the DNA sequence data. Toxicity

studies of the New Zealand C. malayensis isolates (as determined by intraperitoneal (i.p.) injection of

mice) resulted in an LD50 of 80.0 mg/kg for isolate NLD 12 (95% CI: 63.8–101.0 mg/kg) compared to a

Malaysian isolate which exhibited low toxicity, with transient effects at 900 mg/kg, but which resulted

in the enlargement of the test animal’s spleen. Ostreopsis siamensis, a common epiphytic bloom former in

northern New Zealand, was found in association with Coolia at sites across Northland. Densest cell

concentrations were collected from macroalgae throughout the Bay of Islands. Four new isolates of O.

siamensis all produced palytoxin-like compounds (LC–MS analysis). Also isolated from Northland

macroalgae were Amphidinium thermaeum (non-toxic by i.p. injection of mice) and Gambierdiscus cf.

yasumotoi, with the latter producing a putative maitotoxin analogue (MTX-3) as determined by LC–MS.

� 2014 Elsevier B.V. All rights reserved.

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Harmful Algae

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1. Introduction

The closely related dinoflagellate genera Coolia Meunier andOstreopsis J.Schmidt (Order Gonyaulacales F.J.R. Taylor; FamilyOstreopsidaceae Lindemann) have a global distribution. Theepiphytic Coolia is found in coastal regions of warm to tropicaloceans world-wide, including the Mediterranean Sea and theAtlantic coast of France. It attaches to macroalgae, corals andeelgrasses and may also be benthic on sediment surfaces inmangrove habitats (Rhodes et al., 2000; Guiry and Guiry, 2013).Ostreopsis, also predominantly epiphytic, has a current northernboundary of 458390 N (Monti et al., 2007) and southern boundary of458 S (Rhodes, 2011; Parsons et al., 2012), although this range islikely to expand.

There are five currently accepted species within the Coolia

genus: C. monotis Meunier (the type species), C. areolata Ten-Hage,Turquet, Quod & Coute, C. canariensis Fraga, C. malayensis Leaw,

* Corresponding author. Tel.: +64 3 5482319; fax: +64 3 546 9464.

E-mail address: lesley.rhodes@cawthron.org.nz (L. Rhodes).

http://dx.doi.org/10.1016/j.hal.2014.02.004

1568-9883/� 2014 Elsevier B.V. All rights reserved.

P.-T.Lim & Usup, and C. tropicalis Faust. Coolia has homology inthecal construction with Ostreopsis and at one time C. monotis wasincluded within the genus Ostreopsis as O. monotis (Meunier)Lindemann (Besada et al., 1982). It was later named Glenodinium

monotis (Meunier) Biecheler before being accepted as C. monotis

(Guiry and Guiry, 2013).In New Zealand, Coolia was first isolated in 1995 from sea wrack

(comprising foliose and coralline red algae and small brown algae)collected from Ninety Mile Beach, Northland (Rhodes and Thomas,1997). The isolate (held in the Cawthron Institute CultureCollection of Microalgae (CICCM) as CAWD39, but no longermaintained), was identified as C. monotis based on its morphology.A DNA sequence (large sub-unit ribosomal DNA, LSU rDNA) fromthis isolate was also deposited in GenBank (accession numberU92258). This isolate did have strong similarities to C. tropicalis, forexample the sparsely scattered thecal pores. The identification wassupported by the shape and length of the apical pore and shape andarrangement of the apical Plate 10 (wedge-shaped in C. tropicalis)and the precingular Plate 700 (Rhodes and Thomas, 1997). However,a new species from Kota Kinabalu, Malaysia, was described in2010, namely C. malayensis (Leaw et al., 2010). This species fell into

Fig. 1. Map showing sites in Northland, New Zealand, where dinoflagellates were isolated for this study. Samples were collected between 27 January and 29 February 2013.

L. Rhodes et al. / Harmful Algae 34 (2014) 36–41 37

a separate clade to C. monotis and comparison of the LSU rDNAsequence data with CAWD39 indicated that CAWD39 should be re-identified as C. malayensis. (C. malayensis falls within Clade III and C.

monotis within Clade IV; refer Fig. 7, Leaw et al., 2010). C. monotis

was also reported in New Zealand during a 1995–97 survey ofepiphytic dinoflagellates in Northland by Chang et al. (2000), but inlow concentrations and at <25% of the sites sampled and theidentifications were by morphology alone.

New Zealand isolate CAWD39 was toxic to Artemia salina andHaliotis virginea larvae and produced a mono-sulphated polyethertoxin, but was negative for cooliatoxin, a neurotoxin, as deter-mined by HPLC analysis (Rhodes and Thomas, 1997; unpublisheddata). Isolates from Queensland, Australia, described as C. monotis,did produce cooliatoxin (Holmes et al., 1995; Faust and Gulledge,2002), but that classification has been questioned by Mohammad-Noor et al. (2013), who suggested that the isolate was in fact C.

tropicalis. Early reports of cooliatoxin production by C. monotis

suggested that it was strain dependent, but it is now clear thatmorphology alone may not always be sufficient to confirm speciesidentification for Coolia isolates and that DNA sequence analysis isa requirement (Fraga et al., 2008; Leaw et al., 2010). Early reports oftoxic C. monotis should therefore be treated with caution andreferred to as C. cf. monotis if no DNA sequence data is available.

The closely related Ostreopsis is commonly found in the samelocalities as Coolia and in New Zealand is usually found attached tomacroalgal turfs, to red and brown foliose macroalgae growingsub-tidally on rocky shores, or attached to freely floatingmacroalgae (for example, Sargassum sinclairii J.D. Hooker &Harvey). In New Zealand’s Hauraki Gulf, O. siamensis regularlyforms mats which may smother beds of the brown macroalga,Eklonia radiata C. Agardh (Shears and Ross, 2009). The ninecurrently accepted species of Ostreopsis (Guiry and Guiry, 2013) arelikely to be separated into more species in due course. DNAsequence data questions current classifications, but morphologicaldifferences are often infinitesimal, particularly for O. ovata (Pennaet al., 2005, 2010; Sato et al., 2011). In New Zealand, three specieshave been identified from Northland: O. siamensis Schmidt (Chang,

2000; Rhodes et al., 2000; Shears and Ross, 2009), O. lenticularis

Fukuyo and O. ovata Fukuyo (the latter two by morphologicalidentification only; Chang, 2000).

In this study, strains of Coolia and Ostreopsis were isolated fromNorthland, New Zealand (Fig. 1), in January/February 2013 (theaustral summer), and cultured to allow identification to specieslevel based on morphological characteristics and DNA sequencingdata. The toxicity of an isolate of Coolia from Northland, NewZealand was determined and compared with the toxicity of C.

tropicalis and C. malayensis from Malaysia. Putative palytoxin-likecompounds produced by Ostreopsis isolates were determined byliquid chromatography-mass spectrometry (LC–MS).

2. Methods

2.1. Isolation and growth

Dinoflagellate cells were shaken from attached and floatingmacroalgae, corraline turfs and eelgrasses into 50 ml Falcon tubescontaining local sea water. Sea water samples and surfacesediments from the vicinity of mangroves were also collected.Sites were selected throughout coastal Northland, New Zealandwith a focus on the Bay of Islands (Table 1; Fig. 1). Sampling wascarried out in January–February 2013 (late austral summer) at lowtide in rocky areas or by snorkelling at deeper sub-tidal sites.Germanium dioxide was added to the tubes to suppress diatomgrowth (approx. 1% final conc.) and f/2 medium (Guillard, 1975)was added (final conc. 5%) to support dinoflagellate growth.Sample tubes were held under natural sunlight at ambienttemperatures during the day and in an insulated container atnight until reaching the laboratory. Cells were isolated usingmodified pickers (glass tubing drawn out under flame to obtain a>1 mm bore; attached to rubber hose for mouth suction). Cellswere transferred into 24-well tissue culture plates (BectonDickinson, USA) containing f/2 or f/2:sea water (1:1) and grownat 25 �2 8C, 70–100 mmol m�2 s�1 photon irradiance (12:12 h L:D).Selected clonal cultures were deposited in the CICCM.

Table 1New Zealand isolates of Coolia malayensis, Ostreopsis siamensis, Amphidinium thermaeum and Gambierdiscus yasumotoi referred to in this study. The comparative toxicity of

Malaysian isolates of C. malayensis and C. tropicalis (tested by intraperitoneal (i.p.) injection of mice at AgResearch, Hamilton, NZ) is also presented.

Species Site of isolation Lat:Long Strain codea GenBank Access. No. Toxicity

C. malayensis 90 Mile Beach, Northland, NZ 348720 S:1738930 E CAWD39 U92258 Positive Na channel activityb

C. malayensis Rangiputa, Northland, NZ 348870 S:1738380 E CAWD77 KJ422852

C. malayensis Rangaunu Harbour, Northland, NZ 348580 S:1738160 E CAWD154 KJ422855

C. malayensis Rangaunu Harbour, Northland, NZ 348580 S:1738160 E CAWD175 KJ422856

C. malayensis Rangiputa, Northland, NZ 348530 S:1738180 E NLD3

C. malayensis Tapeka Point, Bay of Islands, NZ 358150 S:1748070 E NLD9 KJ422857

C. malayensis Rawhiti Point, Bay of Islands, NZ 358130 S:1748150 E NLD12 KJ422858

C. malayensis Rawhiti Point, Bay of Islands, NZ 358130 S:1748150 E NLD12b KJ422859

C. malayensis Banggi Island, Malaysiac 078170 N:1148120 E K-0972 JX896690 Low toxicity to mice (i.p.)

C. tropicalis Banggi Island, Malaysiac 078170 N:1148120 E K-1156 JX896691 Slow-acting toxicity to mice (i.p.)

O. siamensis Tapeka Point, Bay of Islands, NZ 358150 S:1748070 E CAWD206 KJ422860

O. siamensis Tapeka Point, Bay of Islands, NZ 358150 S:1748070 E CAWD207

O. siamensis Tapeka Point, Bay of Islands, NZ 358150 S:1748070 E CAWD208

A. thermaeum Taupo Bay, Northland, NZ 348600 S:1738430 E CAWD209

G. cf. yasumotoi Te Uenga Bay, Bay of Islands, NZ 358160 S:1748150 E CAWD210 AB859986

G. cf. yasumotoi Te Uenga Bay, Bay of Islands, NZ 358160 S:1748150 E NLD13b

a CAWD strains are held in Cawthron Institute Collection of Micro-algae (CICCM).b Rhodes et al. (2000).c Mohammad-Noor et al. (2013).

L. Rhodes et al. / Harmful Algae 34 (2014) 36–4138

2.2. Identification

2.2.1. Microscopy

Motile cells were photographed using an inverted OlympusIX70 epifluorescence microscope (magnification 600�). Forscanning electron microscopy (SEM), cells were fixed (glutaralde-hyde 3%, formaldehyde 2%, phosphate buffer 0.1 M), then settled orcentrifuged and most of the supernatant removed. Cells weretransferred to a polycarbonate filter and compressed between asecond filter before passing through a graded EtOH series fordehydration and critical point drying. The filters were separatedand the bottom filter mounted on an SEM stub with double-sidedtape for sputter coating with gold, in preparation for SEM (FEIQuanta 200).

2.2.2. DNA sequencing and phylogenetic analyses

Dinoflagellate cultures were centrifuged (10 ml; 542 g, 15 min,RT) and DNA extracted using a PowerSoilTM DNA isolation kit (MoBio Lab. Inc., CA, USA). The D1-D3 fragment of the LSU rDNA wasamplified using primers D1R-F (Scholin et al., 1994) and D3B-R(Nunn et al., 1996). The PCR amplifications were carried out in50 ml reaction volumes containing i-Taq 2x PCR master mix (25 ml;Intron, Gyeonggi-do, Korea), both forward and reverse primers(0.4 mM) and template DNA (ca. 50–150 ng). Thermocyclingconditions included a denaturing step of 95 8C for 4 min; 35cycles of 95 8C for 30 s and 60 8C for 2 min; and a final extensionstep of 72 8C, 10 min. Amplification products were purified(AxyPrep PCR cleanup kits, Axygen, CA, USA) and sequenced (inboth directions using PCR primers) by an external contractor(University of Waikato DNA Sequencing Facility, Hamilton, NZ).Forward and reverse sequences were aligned using Geneious1

v6.0.4 (Drummond et al., 2011) and conflicts resolved by manualinspection. Sequences were aligned with sequences from Coolia

spp. available from GenBank (www.ncbi.nlm.nih.gov) withOstreopsis spp. (isolated in this study and from GenBank; accessionnumbers AF244940, GQ380660, HQ414222, HQ414225) used asout-groups in Geneious1. For subsequent analyses, the D1-D3sequence alignment was truncated to 585 bp. Bayesian analyseswere carried out in Geneious1 using MrBayes 3.1.2 (Huelsenbeckand Ronquist, 2001) using the evolutionary model (general timereversible with gamma-shaped among-site variation, GTR + G).Analyses of alignments were carried out in two simultaneous runswith four chains each for 5.1 � 106 generations, sampling every

100 trees. A 50% majority-rule consensus tree was drawn from thelast 1000 trees.

2.3. Toxin analysis

Dinoflagellate cultures (approx. 0.5–1 L) were centrifuged andpellets extracted and screened for palytoxin and related analoguesusing a quantitative LC-MS/MS method developed at Cawthron(Selwood et al., 2012). The method is more sensitive thanpublished methods, which monitor the intact toxin, as it monitorssubstructures which are generated by oxidative cleavage of vicinaldiol groups present in the intact toxin. This includes and an aminecontaining substructure common to known palytoxins, ovatoxinsand ostreocins.

2.4. Toxicity

Freeze-dried cell pellets from dinoflagellate batch cultures(including Ostreopsis spp., C. malayensis and Amphidinium ther-

maeum from New Zealand and also two Malaysian isolates of C.

tropicalis and C. malayensis; refer Table 3) were extracted withethanol (4 ml), using a Potter-Elvehjem homogeniser and evapo-rated to dryness (40 8C). Residue was taken up in MeOH andaliquots evaporated under N2 then dried overnight in a vacuumdesiccator. The extract was re-suspended in 1% Tween 60 in saline,administered by intraperitoneal injection (i.p.) into Swiss albinomice (18–20 g) at various dose levels, and the LD50 valuesdetermined (OECD, 2006).

3. Results and discussion

The main aim of the study was to determine which Coolia

species were present in the northern, sub-tropical coastal waters ofNew Zealand. Dinoflagellate genera isolated from macroalgae andcultured between January and February 2013 from acrossNorthland, New Zealand (Fig. 1), included Coolia, Ostreopsis,Amphidinium Claperede & Lachmann, Prorocentrum Ehrenbergand Gambierdiscus Adachi & Fukuyo (Rhodes et al., 2013). Both P.

lima (Ehrenberg) F.Stein and P. maculosum M.A. Faust werecultured and identified based on morphological characteristics.Other potentially harmful species included the cyanobacteriaLyngbya majuscula (Dillwyn) Harvey and a species of Anabaena

Bory de Saint-Vincent ex Bornet & Flahault which were isolated

Fig. 2. Phylogenetic analysis of alignment of Coolia partial LSU rDNA sequences (D1-D3 region) using Bayesian analyses and Ostreopsis species as an outgroup. Sequences in

bold are strains isolated in this study. Values at nodes represent Bayesian posterior probability support and the scale is substitutions per site.

L. Rhodes et al. / Harmful Algae 34 (2014) 36–41 39

from surface sediments collected in muddy tidal pools and frommangrove areas. The dominant epiphytic diatom found at all sitessampled was Paralia marina (W. Smith) Heiberg, which reachedhigh monospecific concentrations on some red macroalgae, asdetermined by counts of Lugols Iodine treated samples.

C. malayensis was previously identified in New Zealand as theclosely related and morphologically similar C. monotis (Rhodes andThomas, 1997; Mohammad-Noor et al., 2013) and is a commonepiphyte on seaweeds in New Zealand’s northernmost coastalwaters. New Zealand isolates from Ninety Mile Beach, Northland(CAWD39), Rangaunu Harbour, Northland (CAWD77, 154 and 175)and from aquaculture ponds receiving sea water from Tasman Bay,Nelson, South Island (CAWD98), all fall within the distinct C.

malayensis phylogenetic clade (Leaw et al., 2010; Jeong et al.,2012). An isolate collected from the calcareous green macroalgaHalimeda sp., Cook Islands (CAWD151; Rhodes et al., 2009) alsofalls within the C. malayensis clade. Those strains designated C.

monotis in GenBank (CAWD39) and in the CICCM have now beenreclassified (Table 1). Recently a suite of Coolia species werereported from the central Great Barrier Reef region, Australia,including C. malayensis, C. tropicalis and C. canariensis (Momiglianoet al., 2013). It is likely that more species will be found in northernNew Zealand in the future.

In this study Coolia was isolated from sub-tidal macroalgaethroughout Northland, but in low concentrations. Four isolateswere cultured and all were identified as C. malayensis based on LSU

rDNA sequence data (Fig. 2) and SEMs (Fig. 3). Isolates were codedas NLD 3 (isolated from fronds of Carpophyllum sp., Rangiputa),NLD 9 (isolated from Ceratium sp., Tapeka Point), NLD 12(submitted to CICCM as CAWD214) and 12b (isolated from eelgrass and macroalgae respectively at Kaimarama Bay, RawhitiPoint). There was relatively high genetic diversity in the strainsisolated in this and previous studies, which warrants furtherinvestigation. In particular, strains isolated from Rangaunu(CAWD154 and CAWD175) differed from other strains by up to4% (Fig. 2) and a strain isolated from the Cook Islands (CAWD151)differed from other strains by 2–3%.

Extracts of New Zealand isolates of C. malayensis held in theCICCM were previously tested by i.p. injection of mice withvariable results (Rhodes et al., 2000). One isolate (CAWD39) wasnon-toxic but another (CAWD77) resulted in mouse deathswithin 3 min. An isolate of C. monotis from Spain (CAWD60)tested at that time was non-toxic. In the current study, isolateCAWD214 (isolate NLD 12) had an LD50 by i.p. injection of80.0 mg/kg, with 95% confidence interval between 63.8 and101 mg/kg. The toxin or toxins were slow-acting with deaths upto 5.5 h after injection. The mice became lethargic, with hind limbparalysis. Respiratory rates decreased and death was due torespiratory failure. Isolate NLD 9, at doses of 50, 150 and 450 mg/kg (one mouse per dose-level), resulted in transitory hunching,lethargy and abdominal breathing, but all mice survived inexcellent health.

Fig. 3. Scanning electron micrographs of Coolia malayensis isolated from Northland, New Zealand. (A) Hypothecal plates; (B, C) views of apical pore; (D) cingulum; (E, F) views

of sulcus.

L. Rhodes et al. / Harmful Algae 34 (2014) 36–4140

A Malaysian strain of C. malayensis (submitted for analysis in2011 by Dr Normahwaty Binti Mohammad Noor, Islamic Int. Univ.,Kuala Lumpur, Malaysia) exhibited low toxicity to mice by i.p.injection, with transient effects at 900 mg/kg. However, despite thelow toxicity, adhesions were observed in the peritoneum and thespleen became enlarged (weight >x2 normal; Munday, 2011b). AMalaysian isolate of C. tropicalis (also provided by Dr MohammadNoor), contained a slow-acting toxin which caused the death ofmice when injected at 900 mg/kg (i.e. low toxicity).

The related gonyaulacoid, O. siamensis, is a common bloomformer from the far north of New Zealand to the Hauraki Gulf, oftensmothering macroalgae and causing the death of sea urchins(Rhodes et al., 2006; Shears and Ross, 2009). Isolates may producepalytoxin-like compounds, which are highly toxic to mice via i.p.administration. In this study Ostreopsis was found growingepiphytically on a variety of brown and red macroalgae at 25 of28 coastal sites sampled throughout Northland. Cells wereparticularly concentrated on macroalgae sampled by snorkellingthroughout the Bay of Islands. All isolates were determined as O.siamensis by light and scanning electron microscopy. The LSU rDNAsequence data of three isolates were analysed for confirmation ofidentification and had identical sequences (GenBank accessionnumber KJ422860). Sequences were identical to O. siamensis

strains isolated from Europe (Fig. 2). These three isolates were alsoanalysed by LC–MS and all gave the amine fragment common toknown palytoxins, ovatoxins and ostreocins. In addition, acharacteristic amide fragment common to bishomopalytoxinwas identified from all three isolates, although additional proof-of-structure data is required to be certain of this assignment.

A. thermaeum Dolapsakis et Economou-Amilli, as determined byDNA sequencing (Dolapsakis and Economou-Amilli, 2009; Murrayet al., 2012), was isolated from seaweeds sampled at Taupo Bay, tothe north-west of Whangaroa Harbour (Fig. 1). The trimedsequence (782 bp) was used as a query sequence in a BLASTsearch of the GenBank non-redundant (nr) database. The highesthomology found, using blastn searches, was with an A. thermaeum

strain isolated from Australia (GenBank accession numberJQ394809, coordinates 35–816: E-value = 0.0, percent identi-ty = 100%). It is a new identification for New Zealand. Cultureswere analysed by i.p. injection of mice (dosage 450 mg/kg) and by

haemolysis assay and in both assays were non-toxic. A. thermaeum

is maintained in the CICCM as CAWD209.Large, globular cells, similar morphologically in ventral/dorsal

view to Coolia, were isolated from seaweeds attached to rocks inUenga Bay, east of the township of Russell in the Bay of Islands(Fig. 1). These cells were successfully cultured and identified as G.

cf. yasumotoi by analysis of DNA sequencing data and observationof the thecal plate structure using SEM (Rhodes et al., 2013)(Holmes, 1998; Litaker et al., 2009). LC–MS analysis determinedthe presence of a putative maitotoxin analogue (Rhodes et al.,2013). G. cf. yasumotoi cultures are maintained in the CICCM asCAWD210 and CAW211.

In recent years palytoxin-like compounds have impacted onhuman respiratory health along the Mediterranean coast due toaerosolisation of the toxins produced by O. ovata (Ciminiello et al.,2006). The toxicity of palytoxin-like compounds produced by NewZealand isolates of O. siamensis is equivalent to palytoxin itself(Munday, 2011a). Uptake of palytoxin-like compounds byshellfish, sea urchins and crabs has been fully investigated forNew Zealand commercial species and the levels of toxin present inedible tissue suggests that human poisonings are unlikely (Rhodeset al., 2002, 2006, 2008). Toxicity by oral administration of mice, asopposed to intraperitoneal injection, is also relatively low.However, high concentrations of palytoxin have been recordedin crabs (Yasumoto, 1986; Alcala and Halstead, 1970) and fish(Taniyama et al., 2003), particularly in the Philippines, andadvisories have been given by New Zealand health regulators toremove the ‘gut’ before preparing crabs and crayfish forconsumption. If palytoxin was to become a regulated toxin inthe future there would be economic ramifications for the NZseafood industry and, as a safe-guard, new chemical analyticalmethods have been developed and validated using LC–MS (Sel-wood et al., 2012).

4. Conclusion

C. malayensis is a common dinoflagellate species in NewZealand, occurring throughout the sub-tropical north of NewZealand and as far south as the temperate coastal waters of Nelson.Previous classifications of C. monotis (Rhodes and Thomas, 1997)

L. Rhodes et al. / Harmful Algae 34 (2014) 36–41 41

have now been re-examined and reclassified as C. malayensis.

Extracts of some strains show toxicity to mice by i.p. injection.These strains elicit positive sodium channel activity and are toxicto N2A cells in vitro (Rhodes et al., 2000).

Ostreopsis siamensis is also a common dinoflagellate throughoutNorthland and has been recorded in the temperate waters in thesouth-west of the North Island (Rhodes, 2011; Parsons et al., 2012).The Ostreopsidaceae are commonly found in association withAmphidinium, Prorocentrum and Gambierdiscus. Northland is noexception and new observations of both A. thermaeum andGambierdiscus cf. yasumotoi were made during this study (Rhodeset al., 2013).

Acknowledgements

Sincere thanks to Des and Jan Adams for sample collection fromtheir boat while in the Bay of Islands, to Krystyna Ponikla, thecurator of the Cawthron Institute Culture Collection of Microalgae(CICCM) and Aaron Quarterman for designing Fig. 3. A specialthanks to Doug Hopcroft, Institute of Molecular Biosciences,Massey University, Palmerston North, for his scanning electronmicroscopy expertise. Funding from the Ministry of Business,Innovation and Employment, New Zealand (Contract No.CAWXO703) and a Postdoctoral fellowship (Beatriu de Pinos–Marie Curie COFUND programme, Autonomous Government ofCatalonia) supported this research.[SS]

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