Susanna A. Wood

Sources of edaphic cyanobacterial diversity in the Dry Valleys of Eastern Antarctica.

Cyanobacteria are major components of Antarctic Dry Valley ecosystems. Their occurrence in lakes and ponds is well documented, however, less is known about their distribution in edaphic environments. There has been considerable debate about the contribution of aquatic organic matter derived largely from cyanobacteria to terrestrial ecosystems. In this study, automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity in a range of soil environments within the Miers and Beacon Valleys. These data were used to elucidate the input of aquatic cyanobacteria to soil communities. Thirty-eight samples were collected from a variety of soil environments including dry and moist soils, hypoliths and lake and hydroterrestrial microbial mats. The results from the ARISA and 16S rRNA clone library analysis demonstrated that diverse cyanobacterial communities exist within the mineral soils of the Miers Valley. The soil samples from Beacon Valley were depauparate in cyanobacterial signals. Within Miers Valley, significant portions (29%–58%) of ARISA fragment lengths found in aquatic cyanobacterial mats were also present in soil and hypolith samples, indicating that lacustrine and hydroterrestrial cyanobacteria play a significant role in structuring soil communities. The influence of abiotic variables on the community structure of soil samples was assessed using BEST analysis. The results of BEST analysis of samples from within Miers Valley showed that total percentage of carbon content was the most important variable in explaining differences in cyanobacterial community structure. The BEST analyses indicated that four elements contributed significantly to species compositional differences between valleys. We suggest that the complete absence of lakes or ponds from Beacon Valley is a contributing factor to the low cyanobacterial component of these soils.

High Levels of Structural Diversity Observed in Microcystins from Microcystis CAWBG11 and Characterization of Six New Microcystin Congeners

Microcystins (MCs) are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Differences in the coding of the non-ribosomal peptide synthetase/polyketide synthase enzyme complex responsible for microcystin production have resulted in more than 100 microcystin variants being reported to date. The microcystin diversity of Microcystis CAWBG11 was investigated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-mass spectrometry. This revealed that CAWBG11 simultaneously produced 21 known microcystins and six new congeners: [Asp3] MC-RA, [Asp3] MC-RAba, [Asp3] MC-FA, [Asp3] MC-WA, MC-FAba and MC-FL. The new congeners were putatively characterized by tandem mass spectrometry and chemical derivatization. A survey of the microcystin congeners produced by 49 cyanobacterial strains documented in scientific literature showed that cyanobacteria generally produce four microcystin congeners, but strains which produce up to 47 microcystin congeners have been reported. Microcystis CAWBG11 (which produces at least 27 congeners) was positioned in the top ten percentile of the strains surveyed, and showed fluidity of the amino acids incorporated into both position two and position four.

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.

FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.

Switching toxin production on and off: intermittent microcystin synthesis in a Microcystis bloom

Toxic cyanobacterial blooms are increasing in prevalence. Microcystins are the most commonly produced cyanotoxin. Despite extensive research the variables regulating microcystin production remain unclear. Using a RT-QPCR assay that allowed the precise measurement of mcyE transcriptional gene expression and an ELISA that enabled small changes in total microcystin concentrations to be monitored, we demonstrate for the first time that microcystin production is not always constitutive and that significant up- and downregulation in microcystin synthesis can occur on time scales of 2-6 h. Samples were collected over 3 days from a small eutrophic lake during a dense microcystin-producing Microcystis bloom. McyE gene transcripts were detected in only four out of 14 samples. Vicissitudes in both microcystin quotas and extracellular microcystin levels corresponded with changes in mcyE expression. During the period of exalted microcystin synthesis Microcystis sp. cell concentrations increased from 70 000 cells ml(-1) to 4 000 000 cells ml(-1) . These data provide compelling evidence that changes in Microcystis cell concentrations influence microcystin production.

Widespread Distribution and Identification of Eight Novel Microcystins in Antarctic Cyanobacterial Mats

ABSTRACT The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [d-Asp3] MC-RR and [d-Asp3] MC-LR containing glycine [Gly1] rather than alanine and combinations of homoarginine [hAr2] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda5] substitutions. Nostoc sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing Nostoc sp. from multiple samples.

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.

Polyphasic assessment of fresh‐water benthic mat‐forming cyanobacteria isolated from New Zealand

Mat-forming benthic cyanobacteria are widespread throughout New Zealand rivers, and their ingestion has been linked to animal poisonings. In this study, potentially toxic benthic cyanobacterial proliferations were collected from 21 rivers and lakes throughout New Zealand. Each environmental sample was screened for anatoxins using liquid chromatography-MS (LC-MS). Thirty-six cyanobacterial strains were isolated and cultured from these samples. A polyphasic approach was used to identify each isolate; this included genotypic analyses [16S rRNA gene sequences and intergenic spacer (ITS)] and morphological characterization. Each culture was analysed for anatoxins using LC-MS and screened for microcystin production potential using targeted PCR. The morphospecies Phormidium autumnale was found to be the dominant cyanobacterium in mat samples. Polyphasic analyses revealed multiple slight morphological variants within the P. autumnale clade and highlighted the difficulties in identifying Oscillatoriaceae. Only one morphospecies (comprising the two strains CYN52 and CYN53) of P. autumnale was found to produce anatoxins. These strains formed their own clade based on partial 16S rRNA gene sequences. These data indicate that benthic P. autumnale mats are composed of multiple morphospecies and toxin production is dependent on the presence of toxin-producing genotypes. Further cyanobacteria are also characterized, including Phormidium murrayi, which was identified for the first time outside of Antarctica.

First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh

Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supplys pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g⁻¹ dry weight in the lake sample and 119.4 μg g⁻¹ dry weight or 1.3 pg cell⁻¹ in UCFS10. This is the first confirmation of a saxitoxin-producing species in New Zealand and the first report of saxitoxin production by a species of Scytonema.

Molecular genetic tools for environmental monitoring of New Zealand's aquatic habitats, past, present and the future

The assessment of biological samples is critical for measuring the ‘health’ of New Zealand aquatic environments. Analysis of these samples commonly requires species identification and enumeration, which usually involves microscopy or microbiological methods. These techniques can be time-consuming, laborious, and are dependent on taxonomic expertise. Recent advances in molecular methods provide promising tools for assessing environmental samples. A range of molecular techniques are now used in New Zealand including: fluorescent in situ hybridisation; automated ribosomal intergenic spacer analysis; quantitative polymerase chain reaction; and, most recently, next-generation sequencing. The organisms (or targets) and environments monitored are equally diverse, ranging from cyanobacteria, rotifers and invasive fish in lakes, to macroinvertebrates, and biofilm communities in rivers, to bacteria, micro- and macro-algae and invertebrates in marine ecosystems. Despite research and validation demonstrating their potential, the application of these tools by monitoring agencies has been limited. Legislative requirements, costs, and a reluctance to change methodologies, are the most likely reasons for this. This review examines molecular tools that have been previously or are currently used for monitoring aquatic environments in New Zealand, and explores how these, and new techniques, may be applied in the future.