Development and field assessment of a quantitative PCR for the detection and enumeration of the noxious bloom-former Anabaena planktonica

Anabaena planktonica is a harmful, bloom-forming freshwater cyanobacterium, which has arrived recently in New Zealand. In the short time since its incursion (<10 yr), A. planktonica has spread rapidly throughout lakes in the North Island. To date, the identification and enumeration of A. planktonica has been undertaken using light microscopy. There is an urgent demand for a highly sensitive and specific quantitative detection method that can be combined with a high sample processing capability in order to increase sampling frequency. In this study, we sequenced 36 cyanobacterial 16S rRNA genes (partial), complete intergenic transcribed spacers (ITS), and 23S rRNA genes (partial) of fresh-water cyanobacteria found in New Zealand. The sequences were used to develop an A. Planktonica specific TaqMan QPCR assay targeting the long ITS1-L and the 5´ terminus of the 23S rRNA gene. The QPCR method was linear (R2 = 0.999) over seven orders of magnitude with a lower end sensitivity of approximately five A. planktonica cells in the presence of exogenous DNA. The quantitative PCR (QPCR) method was used to assess the spatial distribution and seasonal population dynamics of A. planktonica from the Lower Karori Reservoir (Wellington, New Zealand) over a five-month period. The QPCR results were compared directly to microscopic cell counts and found to correlate significantly (95% confidence level) under both bloom and non-bloom conditions. The current QPCR assay will be an invaluable tool for routine monitoring programs and in research investigating environmental factors that regulate the population dynamics and the blooming of A. planktonica

No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida)

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography—mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely

Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria)

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring

Increasing Microcystis cell density enhances microcystin synthesis: a mesocosm study

An experimental protocol using mesocosms was established to study the effect of Microcystis sp. cell abundance on microcystin production. The mesocosms (55 L) were set up in a shallow eutrophic lake and received either no (control), low (to simulate a moderate surface accumulation), or high (to simulate a dense surface scum) concentrations of Microcystis sp. cells collected from the lake water adjacent to the mesocosms. In the low- and high-cell addition mesocosms (2 replicates of each), the initial addition of Microcystis sp. cells doubled the starting cell abundance from 500 000 to 1 000 000 cells mL⁻¹, but there was no detectable effect on microcystin quotas. Two further cell additions were made to the high-cell addition mesocosms after 60 and 120 min, increasing densities to 2 900 000 and 7 000 000 cells mL-1, respectively. Both additions resulted in marked increases in microcystin quotas from 0.1 pg cell-1 to 0.60 and 1.38 pg cell⁻¹, respectively, over the 240 min period. Extracellular microcystins accounted for <12% of the total microcystin load throughout the whole experiment. The results of this study indicate a relationship between Microcystis cell abundance and/or mutually correlated environmental parameters and microcystin synthesis

Hindcasting cyanobacterial communities in Lake Okaro with germination experiments and genetic analyses

Cyanobacterial blooms are becoming increasingly prevalent worldwide. Sparse historic phytoplankton records often result in uncertainty as to whether bloom-forming species have always been present and are proliferating in response to eutrophication or climate change, or if there has been a succession of new arrivals through recent history. This study evaluated the relative efficacies of germination experiments and automated rRNA intergenic spacer analysis (ARISA) assays in identifying cyanobacteria in a sediment core and thus reconstructing the historical composition of cyanobacterial communities. A core (360 mm in depth) was taken in the central, undisturbed basin of Lake Okaro, New Zealand, a lake with a rapid advance of eutrophication and increasing cyanobacteria populations. The core incorporated a tephra from an 1886 volcanic eruption that served to delineate recent sediment deposition. ARISA and germination experiments successfully detected akinete-forming nostocaleans in sediment dating 120 bp and showed little change in Nostocales species structure over this time scale. Species that had not previously been documented in the lake were identified including Aphanizomenon issatschenkoi, a potent anatoxin-a producer. The historic composition of Chrococcales and Oscillatoriales was more difficult to reconstruct, potentially due to the relatively rapid degradation of vegetative cells within sediment

Further characterization of glycine-containing microcystins from the McMurdo Dry Valleys of Antarctica

Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 D-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species

Tetrodotoxin Concentrations in Pleurobranchaea maculata: Temporal, Spatial and Individual Variability from New Zealand Populations

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg−1) and maximum (1414.0 mg kg−1) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg−1 in June 2010 to 17.5 mg kg−1 by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June–August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected

Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration.

PurposeIntravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model.MethodsC3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina.ResultsIn vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis.ConclusionsIntravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration

Increasing Microcystis cell density enhances microcystin synthesis: a mesocosm study

An experimental protocol using mesocosms was established to study the effect of Microcystis sp. cell abundance on microcystin production. The mesocosms (55 L) were set up in a shallow eutrophic lake and received either no (control), low (to simulate a moderate surface accumulation), or high (to simulate a dense surface scum) concentrations of Microcystis sp. cells collected from the lake water adjacent to the mesocosms. In the low- and high-cell addition mesocosms (2 replicates of each), the initial addition of Microcystis sp. cells doubled the starting cell abundance from 500 000 to 1 000 000 cells mL-1, but there was no detectable effect on microcystin quotas. Two further cell additions were made to the high-cell addition mesocosms after 60 and 120 min, increasing densities to 2 900 000 and 7 000 000 cells mL-1, respectively. Both additions resulted in marked increases in microcystin quotas from 0.1 pg cell-1 to 0.60 and 1.38 pg cell-1, respectively, over the 240 min period. Extracellular microcystins accounted for &lt;12% of the total microcystin load throughout the whole experiment. The results of this study indicate a relationship between Microcystis cell abundance and/or mutually correlated environmental parameters and microcystin synthesis