Cyanobacteria (blue-green algae) produce a diverse array of toxic or otherwise bioactive metabolites. These allelochemicals may also play a role in defense against potential predators and grazers, particularly aquatic invertebrates and their larvae, including mosquitoes. Compounds derived from cyanobacteria collected from the Florida Everglades and other Florida waterways were investigated as insecticides against the mosquito Aedes aegypti, a vector of dengue and yellow fever. Screening of cyanobacterial biomass revealed several strains that exhibited mosquito larvicidal activity. Guided via bioassay guided fractionation, a non-polar compound from Leptolyngbya sp. 21-9-3 was found to be the most active component. Characterization revealed the prospective compound to be a monounsaturated fatty acid with the molecular formula C16H30O2. This is the first evidence of mosquito larvicidal activity for this particular fatty acid. With larvicidal becoming more prevalent, fatty acids should be explored for future mosquito control strategies.

Black band disease (BBD) is a cyanobacterial dominated pathogenic consortium that affects corals worldwide. Recently two cyanobacteria (Oscillatoria strains 101-1 and 100-1) were isolated into culture from BBD. The aim of this study was to characterize the strains and assess their role in BBD pathogenesis. Light, scanning electron and transmission electron microscopy, coupled with 16S rRNA gene sequencing, were used for taxonomic characterization. Cyanotoxin production was assessed by enzyme-linked immunosorbent assay. Toxin identification was performed by high performance liquid chromatography. The ability of the strains to initiate BBD was tested on host coral fragments of Siderastrea siderea and Diploria strigosa under controlled laboratory conditions. Results showed that both Oscillatoria sp. strains caused infection that resulted in complete lysis of coral tissue. Both strains produced a cyanotoxin, identified as microcystin-LR, with production affected by different environmental factors. This study provides evidence that BBD Oscillatoria is a key component in BBD pathogenicity.

β-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid associated with human neurodegenerative diseases. The content of BMAA in cyanobacteria is modulated by nitrogen in laboratory cultures. In order to evaluate the potential for human exposure, the nitrogen modulation of BMAA content needed verification in a natural environment. In accordance with laboratory culture studies, data presented in this study show that combined nitrogen was the most significant modulator of both cellular microcystin (MC) and BMAA content in phytoplankton in an environmental cyanobacterial bloom. While BMAA is produced upon nitrogen deprivation, MC is only produced at a specific nitrogen threshold where the rate of increase of nitrogen in the cell exceeds the carbon fixation rate. As BMAA and MC were detected in phytoplankton sourced from the Hartbeespoort Dam reservoir, the transfer of these cyanotoxins to organisms of higher trophic levels was investigated. Both BMAA and MC were detected at high concentrations in the liver and muscle tissue of fish sourced from the Hartbeespoort Dam reservoir indicating that consumption of fish from this reservoir constitutes a serious risk of exposure to cyanotoxins. In addition to the dietary exposure route to BMAA, two recent studies reported a correlation between Amyotrophic Lateral Sclerosis (ALS) incidence and the potential for aerosol exposure to cyanobacteria. With the absence of any evidence of the systemic distribution of BMAA following inhalation, an evaluation of the potential exposure risk associated with living in close proximity to this reservoir was deemed premature. A laboratory experiment investigating the effect and systemic fate of inhaled aerosolised BMAA was therefore conducted in order to determine the feasibility of inhalation as a potential BMAA exposure route. Data from the rat inhalation exposure study, however, showed that in rats BMAA inhalation may not constitute a significant mechanism of toxicity at environmental BMAA levels.

β-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid associated with human neurodegenerative disease. Due to the cosmopolitan nature of cyanobacteria, detection of BMAA in cyanobacteria has caused concerns about human exposure risk. This study was therefore based on the hypothesis that BMAA poses a health risk to humans either by direct ingestion or by indirect exposure to BMAA from a cyanobacterial source via a freshwater food chain. A validated gas chromatography-mass spectrometry (GC-MS) BMAA analysis method and a confirmatory liquid chromatography-mass spectrometry (LC-MS) method, with improved sensitivity, were developed in addition to a LC-MS/MS method for analyte confirmation. These methods were used to quantify BMAA in South African cyanobacteria, isolated from various potable water reservoirs. The majority of the isolates tested, contained BMAA. Possible human exposure by direct consumption of BMAA released from cyanobacterial blooms was investigated by the development of a robust solid phase extraction (SPE) method used for BMAA concentration and quantification in raw and treated tap water. Despite the use of the SPE method that facilitated the concentration of BMAA from large quantities of water, no free dissolved BMAA was detected in raw or processed fresh water. The fate of exogenous BMAA was therefore investigated firstly by evaluating the efficacy of standard water treatment processes employed in South Africa and secondly by investigating the possibility of BMAA bioaccumulation and biomagnification in aquatic food chains. Standard water treatment processes proved highly efficient at removing free dissolved BMAA, explaining the absence of BMAA in treated tap water. However, the cause of the BMAA absence in raw potable water remained unknown. Uptake of BMAA by model aquatic organisms was investigated in controlled experiments. BMAA uptake was documented in both Ceratophyllum demersum and Daphnia magna, however, BMAA-protein association and biomagnification were not observed in D. magna. BMAA had an inhibitory effect on the oxidative stress enzyme acitivties of both organisms tested (as well as human S9 extracts), resulting in accumulation of detrimental reactive oxygen species (ROS) in the cells. Exposure of crop plants to BMAA in controlled experiments resulted in BMAA uptake, protein association, and subsequent inhibition of the antioxidative enzyme activities. However, BMAA was detected in neither free nor protein-associated form in natural crop plants irrigated with known BMAA-containing bloom water. Post-mortem liver samples of Clarias gariepinus (Catfish) and Crocodylus niloticus (Crocodile), from a natural fresh water ecosystem that experienced frequent cyanobacterial blooms, contained both free and protein-associated BMAA. Higher BMAA concentrations were found in crocodile liver samples compared to fish liver samples, strongly suggesting biomagnification from one trophic level to the next. BMAA concentrations corresponded to crocodile age. This is the first report of bioaccumulation and biomagnification in two trophic levels in a fresh water ecosystem. These findings strongly suggest possible human exposure via aquatic food chains of cyanobacterial origin. Direct BMAA exposure via drinking water is not plausible due to the efficiency of standard water treatment processes to remove BMAA. The use of raw water for agricultural and recreational use, however, remains a problem. The development of management strategies as well as daily tolerable levels for BMAA is urgently required.

Cyanobacterial blooms, such as those recently reported in Great Slave Lake (GSL, NWT), have sparked concern over the occurrence of toxic blooms in the North. This study investigated past and present incidences of cyanobacteria in lakes above latitude 60° N. The abundance of the toxin (microcystin) gene mcyE, as well as genes common to all cyanobacteria (16S rRNA) and bacteria (glnA) were quantified from lake sediment cores using ddPCR. Individual colony isolates from a surface bloom in Yellowknife Bay (GSL) in August 2015 were amplified and identified as non-toxigenic Dolichospermum lemmermannii. Very low levels of microcystin genes were detected through the sediment archives (over ~100-150 yr) of GSL and other lakes, as well as in the plankton of GSL. While recent increases in mcyE were not observed, an increase in the cyanobacterial 16S rRNA and glnA genes was seen through time. In the high Arctic Meretta Lake, gene abundance profiles reflected the effects of past eutrophication and recovery.

Yersinia pseudotuberculosis is a mammalian enteropathogen and is the direct ancestor of Y. pestis, the causative agent of the plague. For its pathogenicity, Y. pseudotuberculosis harbours a 70 kb virulence plasmid which encodes the components of the type three secretion system (T3SS) and effector proteins. These effectors serve to evade the host immune system and induce apoptosis of mammalian cells. Consistent with many Gram-negative bacteria, Yersinia facilitate cell: cell signalling through the production and sensing of N-acylhomoserine lactones (AHLs), which functions to mediate the expression of downstream target genes. This cell-cell communication is known as quorum sensing (QS) and is facilitated by two LuxI/R-type systems in Y. pseudotuberculosis: YtbI/R and YpsI/R, and several AHL molecules. Behaviours under QS control include motility, biofilm formation, clumping and the regulation of the T3SS. Recently, QS was reported to repress the T3SS whilst the T3SS attenuated biofilm formation on Caenorhabditis elegans. Colonising both the soil/water environment and the mammalian gut, Y. pseudotuberculosis exhibits a biphasic lifestyle whereby it exerts strict temperature-dependent control over the expression of pYV-encoded genes. The switch between these two lifestyles is govered by a pair of virulence regulators: LcrF is a transcriptional activator that targets pYV-encoded genes and is key for the assembly of the T3SS. Conversely, YmoA is a histone-like protein that represses transcription of lcrF through chromatin compaction. Considering the repression of the T3SS by QS, this study set out to investigate whether this regulation is mediated by a relationship between QS and LcrF/YmoA. By using chromosomal promoter:lux fusions, QS was identified to be an activator of YmoA at both 22oC and 37oC whilst a regulatory relationship between QS and LcrF was also identified. To investigate these links further, AHL profiling of the lcrF and ymoA mutants identified YmoA as a repressor of AHL biosynthesis whilst a very subtle repression was observed in ΔlcrF, suggesting that LcrF may influence AHL synthesis indirectly. Assessing the impact of LcrF and YmoA on the QS-mediated phenotypes of Yop secretion, biofilm formation and motility extended these observations. LcrF had no effect on any of the phenotypes examined supporting the hypothesis of either an indirect mode of regulation, or no regulation at all. In contrast, YmoA influenced both motility and biofilm formation. A decreased motility of ΔymoA was observed on both semi-solid agar and in liquid whereby both the speed and the percentage of motile cells was altered. This suggests an activating role of YmoA on motility. Interestingly, QS is known to repress motility therefore it is likely that YmoA-regulation of motility occurs irrespective of QS. Comparable to that of the QS synthase mutant (ΔypsI/ytbI), biofilm was attenuated in ΔymoA yet restored when cells were cured of the virulence plasmid supporting the hypothesis that the type three-secretion injectisome disrupts biofilm formation. This attenuation of biofilm formation in ΔymoA, in conjunction with the activation of ymoA by QS, led to the hypothesis that the repression of the T3SS by QS works through YmoA. Considering these results, evidence for an interaction between QS and virulence regulators LcrF and YmoA has been confirmed. We propose a model whereby YmoA is the missing link in the QS-mediated repression of the T3SS. Activation of YmoA by QS leads to increased repression of lcrF and subsequently, of the T3SS resulting in the de-repression of this system in the absence of QS.

Harmful cyanobacterial blooms have been a growing concern as global climate change and eutrophication of lakes, rivers, and oceans continually push conditions to favor cyanobacteria over other phytoplankton. Two studies were conducted assessing the impacts of hyper-eutrophication on phytoplankton communities, and phosphorous saturation in the sediments. Excess nutrients available to phytoplankton resulted in dominant cyanobacteria, and predictability of growth, by nutrient limitation, becoming drastically diminished. Sediments were observed to be fully phosphorus saturated, preventing the sequestration of excess phosphorus, and providing a consistent source of phosphorus throughout each season. Extreme saturation of nutrients reduces the predictability of systems and perpetuates the cycles of nutrient release, fueled by the growth and decay of harmful cyanobacterial blooms.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18477号 / 農博第2077号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4861(農学部図書室) / 31355 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Cyanobacterial algal bloom is a major water quality issue in inland lakes, reservoirs, and estuarine environments because of its scum and bad odor forming and toxin producing abilities. Health risks from cyanobacterial toxin can vary from skin irritations to fever, intestinal problems, and neurological disorders. Terminations of blooms also cause oxygen depletion leading to hypoxia and widespread fish kills. Adding to the problem, many species of cyanobacteria produce odorous compounds such as geosmin and 2-methylisoborneol (MIB) that cause “earthy-muddy” and “musty” odor in drinking water, which is also a serious issue in aquaculture and drinking water industry. Therefore continuous monitoring of cyanobacterial presence in recreational water bodies, surface drinking water sources, and water bodies dedicated for aquaculture is highly required for their early detection and subsequent issuance of a health warning and reducing the economic loss. Remote sensing techniques offers the capability of identifying and monitoring cyanobacterial blooms in a synoptic scale. Over the years, the scientific community has focused on developing methods to quantify cyanobacterial biomass using phycocyanin,an accessory photosynthetic pigment, as a marker pigment. However, because of the confounding influence of chlorophyll-a and other photo pigments, remote retrieval of phycocyanin signal from turbid productive water has been a difficult task. This dissertation analyzes the potential of remote sensing techniques and develops empirical and quasi-analytical algorithms to isolate the phycocyanin signal from the remote sensing reflectance data using a set of radiative transfer equations and retrieves phycocyanin concentration in the water bodies. An extensive dataset, consisting of in situ radiometric measurements, absorption measurements of phytoplankton, colored dissolved organic matter, detritus, and pigment concentration, was used to optimize the algorithms. Validations of all algorithms were also performed using an independent dataset and errors and uncertainties from the algorithms were discussed. Despite the simplicity, an empirical model produced highest accuracy of phycocyanin retrieval, whereas, the newly developed quasi-analytical phycocyanin algorithm performed better than the existing semi-analytical algorithm. Results show that remote sensing techniques can be used to quantify cyanobacterial phycocyanin abundance in turbid and hypereutrophic waters.