Kirsty F. Smith

Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests.

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1–V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide.

Phylogeography of Ostreopsis along West Pacific Coast, with Special Reference to a Novel Clade from Japan

Background A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. Methods/Principal Finding In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8–D10 region of the nuclear large subunit rDNA (D8–D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1–5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8–D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8–D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. Conclusions/Significance This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8–D10 marker will be suitable for these purposes.

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell(-1)). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.

Dinoflagellate Vulcanodinium rugosum identified as the causative organism of pinnatoxins in Australia, New Zealand and Japan

Rhodes L., Smith K., Selwood A., McNabb P., Munday R., Suda S., Molenaar S. and Hallegraeff G. 2011. Dinoflagellate Vulcanodinium rugosum identified as the causative organism of pinnatoxins in Australia, New Zealand and Japan. Phycologia 50: 624–628. DOI: 10.2216/11-19.1 The producers of pinnatoxins E and F were first identified through systematic light microscopy-mass spectrometry (LC-MS) screening of New Zealand (NZ) dinoflagellate isolates. Pinnatoxins E, F and G were subsequently detected for South Australian (AUS) isolates, and a pinnatoxin G producer was recently identified from Okinawa, Japan. The NZ, AUS and Japanese isolates appear morphologically identical to Mediterranean field samples recently described as Vulcanodinium rugosum. Motile forms (24–32 µm long, 20–30 µm wide) have a wide, deeply incised cingulum displaced by 17% of the cell length and a deep sulcus. Photosynthetic motile cells have the same distinctive apical pore complex with raised collar and short canal plate. The reticulate thecal ornamentation resembles that of Bysmatrum because the three intercalary plates are connected. The posterior sulcal plate Sp does not touch the cingulum (as in Bysmatrum, Pentapharsodinium and Ensiculifera). The plate formula for the NZ, AUS and Japanese isolates is Po, X, 4′, 3a, 7″, 6c (5c + T), 5s, 5′″, 2″″. The dominant non-motile life-cycle stage comprises clusters of brown to black, spherical, unornamented, non-calcareous division cells (30–35 µm diameter). Genetically these isolates fall within the Peridiniales, closely related to the calciodinellid genera, Pentapharsodinium, Ensiculifera and Scrippsiella. The large subunit (LSU) rDNA and internal transcribed spacer (ITS) sequences exhibit sequence differences compared to each other and to the French field material (96–97% LSU and 84–88% ITS similarity, respectively). This variation and pinnatoxin profile differences suggest a possible species complex.

Maintenance of cyanotoxin production by cryopreserved cyanobacteria in the New Zealand culture collection

Abstract A culture collection of freshwater planktonic and benthic cyanobacteria collected from sites across New Zealand has been established at the Cawthron Institute, Nelson, New Zealand. Limited resources led to uncertainty regarding the long‐term maintenance of this collection. The present study demonstrates cryopreservation to be a viable method for long‐term storage of cyanobacteria. Seventeen of 20 strains evaluated were successfully cryopreserved using the permeating cryopreservation agent dimethyl sulfoxide (Me2SO), at a final concentration of 15% (v/v). Cyanotoxin analysis was undertaken on selected strains known to produce microcystins, nodularin, anatoxin‐a, and saxitoxins. All strains retained their ability to produce these toxins following cryopreservation.

The first report of Vulcanodinium rugosum (Dinophyceae) from the South China Sea with a focus on the life cycle

An individual non-motile (NM) cell was isolated from a surface sediment sample collected in Guangxi, China and subsequently established as a dinoflagellate strain in culture. The motile cells are 22.5–32.5 µm long and 20.0–30.0 µm wide, with a plate formula of Po, X, 4′, 3a, 7″, 6c, 5(?)s, 5″′, 2″″, fitting the description of Vulcanodinium rugosum. The Chinese strain shares 99.8%, 97.4% and 96.7% similarity (LSU sequence) with those from Australasia, France and Japan. Asexual division of V. rugosum takes place either in the thecate motile stage or within a NM division cell. Motile cells divided by binary fission inside the parent cell and transformed to NM division cells within 24 h. The NM cell underwent one to three consecutive divisions within the parent wall. The divisions were not always synchronous and neither was the release of motile cells from the NM cells. It generally took 4 to 6 days for the NM cells to complete one division. NM cells survived for 1 month at 4 °C in the dark, suggesting that they might play an important role in species dispersal. A novel pinnatoxin was detected at 20 pg cell−1 and no other known pinnatoxins (A–G) were detected.

Detection of Azadinium poporum in New Zealand: the use of molecular tools to assist with species isolations

A real-time PCR assay for the detection of species from the genera Azadinium and Amphidoma (family Amphidomataceae) was developed in order to screen field samples and to aid in the isolation of azaspiracid (AZA)-producing dinoflagellates. The assay was highly specific and sensitive and allowed the rapid detection of target species. Samples collected as part of the New Zealand Marine Phytoplankton Monitoring Programme were analysed using the Amphidomataceae real-time PCR assay. Azadinium poporum was detected in New Zealand for the first time, and a culture was successfully established. Extracts of this isolate proved to be of low toxicity to mice and did not contain AZA-1, -2 or -3. Field samples will continue to be screened with the aim of identifying AZA-producing species. The Amphidomataceae real-time PCR assay will be a useful tool for monitoring programmes and taxonomic surveys worldwide.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

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.

New scenario for speciation in the benthic dinoflagellate genus Coolia (Dinophyceae)

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.

Shifts and stasis in marine HAB monitoring in New Zealand

This review article outlines harmful algal bloom (HAB) monitoring practices in New Zealand and highlights the shift from light microscope (LM)-based identification and quantification of the early 1990s to the use of molecular tools to support the HAB monitoring programmes two decades later. Published research and available client information from the monitoring programmes have been reviewed; HAB events and programme changes are highlighted. The current HAB monitoring practices allow for rapid determination of potential biotoxin issues for the shellfish industry and of potential ichthyotoxic events for finfish farmers. The use of molecular tools, including quantitative PCR, has improved risk assessments for those HAB species that are difficult to differentiate to species level using LM. This has enabled rapid feedback to aquaculture managers during HAB events. Tests for biotoxins in flesh remain the regulatory tools for commercially harvested shellfish, but this is supported by the weekly phytoplankton monitoring data. Recreational (non-commercial) shellfish harvesting and commercial finfish aquaculture rely solely on phytoplankton monitoring to assess the biotoxin risk. HAB monitoring in New Zealand continues to maintain internationally recognised standards, and the government-funded research programmes feed the latest knowledge and technical methods into the programmes. The early dependence on light microscopy continues but is now supported by molecular tools, with a view to employing multi-species detection systems in the future. The traditional mouse bioassay test has been fully replaced by chemical tests.