Comparative studies on physiological responses to phosphorus in two phenotypes of bloom-forming Microcystis

Toxic Microcystis blooms frequently occur in eutrophic water bodies and exist in the form of colonial and unicellular cells. In order to understand the mechanism of Microcystis dominance in freshwater bodies, the physiological and biochemical responses of unicellular ( 4 strains) and colonial ( 4 strains) Microcystis strains to phosphorus ( P) were comparatively studied. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. The growth of four unicellular and one small colonial Microcystis strain was significantly inhibited at a P concentration of 0.2 mg l - 1; however, that of the large colonial Microcystis strains was not inhibited. The results of phosphate uptake experiments conducted using P- starved cells indicated that the colonial strains had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. Alkaline phosphatase activity in the unicellular strains was significantly induced by low P concentrations. Under P- limited conditions, the oxygen evolution rate, Fv/ Fm, and ETRmax were lower in unicellular strains than in colonial strains. These findings may shed light on the mechanism by which colonial Microcystis strains have an advantage with regard to dominance and persistence in fluctuating P conditions.Toxic Microcystis blooms frequently occur in eutrophic water bodies and exist in the form of colonial and unicellular cells. In order to understand the mechanism of Microcystis dominance in freshwater bodies, the physiological and biochemical responses of unicellular ( 4 strains) and colonial ( 4 strains) Microcystis strains to phosphorus ( P) were comparatively studied. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. The growth of four unicellular and one small colonial Microcystis strain was significantly inhibited at a P concentration of 0.2 mg l - 1; however, that of the large colonial Microcystis strains was not inhibited. The results of phosphate uptake experiments conducted using P- starved cells indicated that the colonial strains had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. Alkaline phosphatase activity in the unicellular strains was significantly induced by low P concentrations. Under P- limited conditions, the oxygen evolution rate, Fv/ Fm, and ETRmax were lower in unicellular strains than in colonial strains. These findings may shed light on the mechanism by which colonial Microcystis strains have an advantage with regard to dominance and persistence in fluctuating P conditions

Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria)

Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions.Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions

Physiological comparison between colonial and unicellular forms of Microcystis aeruginosa Kutz. (Cyanobacteria)

In order to gain insight into the bloom sustainment of colonial Microcystis aeruginosa Katz., physiological characterizations were undertaken in this study. Compared with unicellular Microcystis, colonial Microcystis phenotypes exhibited a higher maximum photosynthetic rate (Pm), a higher maximum electron transfer rate (ETRmax), higher phycocyanin content, and a higher affinity for inorganic carbon (K-0.5 DIC <= 8.4 +/- 0.7 mu M) during the growth period monitored in this study. This suggests that photosynthetic efficiency is a dominant physiological adaptation found in colonial Microcystis, thus promoting bloom sustainment. In addition, the high content of soluble and total carbohydrates in colonial Microcystis suggests that this phenotype may possess a higher ability to tolerate enhanced stress conditions when compared to unicellular (noncolonial) phenotypes. Therefore, high photosynthetic activities and high tolerance abilities may explain the bloom sustainment of colonial Microcystis in eutrophic lakes.In order to gain insight into the bloom sustainment of colonial Microcystis aeruginosa Katz., physiological characterizations were undertaken in this study. Compared with unicellular Microcystis, colonial Microcystis phenotypes exhibited a higher maximum photosynthetic rate (Pm), a higher maximum electron transfer rate (ETRmax), higher phycocyanin content, and a higher affinity for inorganic carbon (K-0.5 DIC <= 8.4 +/- 0.7 mu M) during the growth period monitored in this study. This suggests that photosynthetic efficiency is a dominant physiological adaptation found in colonial Microcystis, thus promoting bloom sustainment. In addition, the high content of soluble and total carbohydrates in colonial Microcystis suggests that this phenotype may possess a higher ability to tolerate enhanced stress conditions when compared to unicellular (noncolonial) phenotypes. Therefore, high photosynthetic activities and high tolerance abilities may explain the bloom sustainment of colonial Microcystis in eutrophic lakes

Post-impoundment biomass and composition of phytoplankton in the Yangtze River

Damming, and thus alteration of stream flow, promotes higher phytoplankton populations and encourages algal blooms (density &gt; 10(6) cells L-1) in the Three Gorges Reservoir (TGR). Phytoplankton composition and biomass were studied in the Yangtze River from March 2004 to May 2005. 107 taxa were identified. Diatoms were the dominant group, followed by Chlorophyta and Cyanobacteria. In the Yangtze River, algal abundance varied from 3.13 x 10(3) to 3.83 x 10(6) cells L-1, and algal biomass was in the range of 0.06 to 659 mg C m(-3). Levels of nitrogen, phosphorus and silica did not show consistent longitudinal changes along the river and were not correlated with phytoplankton parameters. Phytoplankton abundance was negatively correlated with main channel discharge (Spearman r = -1.000, P &lt; 0.01). Phytoplankton abundance and biomass in the Yangtze River are mainly determined by the hydrological conditions rather than by nutrient concentrations.Damming, and thus alteration of stream flow, promotes higher phytoplankton populations and encourages algal blooms (density > 10(6) cells L-1) in the Three Gorges Reservoir (TGR). Phytoplankton composition and biomass were studied in the Yangtze River from March 2004 to May 2005. 107 taxa were identified. Diatoms were the dominant group, followed by Chlorophyta and Cyanobacteria. In the Yangtze River, algal abundance varied from 3.13 x 10(3) to 3.83 x 10(6) cells L-1, and algal biomass was in the range of 0.06 to 659 mg C m(-3). Levels of nitrogen, phosphorus and silica did not show consistent longitudinal changes along the river and were not correlated with phytoplankton parameters. Phytoplankton abundance was negatively correlated with main channel discharge (Spearman r = -1.000, P < 0.01). Phytoplankton abundance and biomass in the Yangtze River are mainly determined by the hydrological conditions rather than by nutrient concentrations

Hydrogen peroxide induces apoptotic-like cell death in Microcystis aeruginosa (Chroococcales, Cyanobacteria) in a dose-dependent manner

We investigated the capability of Microcystis aeruginosa to cause apoptosis by pursuing morphological, molecular and physiological characteristics after exposure to H2O2. Microcystis proliferation was only weakly affected after exposure to 150 mu M H2O2 but cell numbers decreased dramatically after exposures of 250 and 325 mu M H2O2. Cells exposed to 250 and 325 mu M H2O2 were examined using transmission electron microscopy, and they exhibited membrane deformation and partial disintegration of thylakoids. Correspondingly, fluorescence imaging of DNA by Hoechst 33342 staining revealed the condensation of nucleoid chromatin. Moreover, cellular injury was concomitant with dramatic decreases in photosynthetic efficiency (ratio of variable fluorescence to maximum fluorescence [Fv/Fm], maximum electron transport rate [ETRmax]) and elevated caspase-3-like activity after exposure of 250 and 325 mu M H2O2. Terminal deoxynucleotidyl transferase Deoxyuridine 5-triphosphate nick end labelling (TUNEL) positive staining appeared in cells exposed to 250 mu M and 325 mu M H2O2, and the percentage staining increased with increasing H2O2 concentration. These data suggested that M. aeruginosa exposed to H2O2 underwent an apoptotic event. Additionally, cells exposed to H2O2 had increased cytoplasmic vacuolation and nontypical DNA laddering. Increased caspase-3-like activity was not inhibited in the presence of the synthetic caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone. Therefore, H2O2 induced apoptotic-like cell death in a dose-dependent manner. Taken together, our results provided a novel mechanism for explaining cyanobacterial bloom dynamics in response to environmental stress. The results also contributed to the understanding of the origin and evolution of programmed cell death.We investigated the capability of Microcystis aeruginosa to cause apoptosis by pursuing morphological, molecular and physiological characteristics after exposure to H2O2. Microcystis proliferation was only weakly affected after exposure to 150 mu M H2O2 but cell numbers decreased dramatically after exposures of 250 and 325 mu M H2O2. Cells exposed to 250 and 325 mu M H2O2 were examined using transmission electron microscopy, and they exhibited membrane deformation and partial disintegration of thylakoids. Correspondingly, fluorescence imaging of DNA by Hoechst 33342 staining revealed the condensation of nucleoid chromatin. Moreover, cellular injury was concomitant with dramatic decreases in photosynthetic efficiency (ratio of variable fluorescence to maximum fluorescence [Fv/Fm], maximum electron transport rate [ETRmax]) and elevated caspase-3-like activity after exposure of 250 and 325 mu M H2O2. Terminal deoxynucleotidyl transferase Deoxyuridine 5-triphosphate nick end labelling (TUNEL) positive staining appeared in cells exposed to 250 mu M and 325 mu M H2O2, and the percentage staining increased with increasing H2O2 concentration. These data suggested that M. aeruginosa exposed to H2O2 underwent an apoptotic event. Additionally, cells exposed to H2O2 had increased cytoplasmic vacuolation and nontypical DNA laddering. Increased caspase-3-like activity was not inhibited in the presence of the synthetic caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone. Therefore, H2O2 induced apoptotic-like cell death in a dose-dependent manner. Taken together, our results provided a novel mechanism for explaining cyanobacterial bloom dynamics in response to environmental stress. The results also contributed to the understanding of the origin and evolution of programmed cell death

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3x10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 mu m in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3x10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 mu m in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium

CyanoNews (Vol. 14, No. 1, August 1998)

CyanoNews was a newsletter that served the cyanobacteriological community from 1985 to 2003, with content provided by readers (sort of a blog before there were blogs). The newsletter reported new findings from the lab, summaries of recent meetings (often provided by graduate students and post-docs entering the field), positions sought or available, life transitions, a compendium of recent cyanobacteria-related articles, and other items of interest to those who study cyanobacteria

Diversité structurelle et fonctionnelle des communautés bactériennes de la phycosphère des cyanobactéries proliférant au sein des écosystèmes lacustres

Potentially toxic cyanobacteria blooms often occur in eutrophic aquatic ecosystems. While many studies have been published on their ecology and toxicity, few have investigated the interactions between cyanobacteria and their associated chimiotrophic bacteria within the phycosphere. This latter is the subject of this thesis. Using both natural ecosystems and laboratory approaches, we show that the structure and composition of bacterial communities (BC) associated with cyanobacteria are different from those that are not under the direct influence of the cyanobacteria. These associated communities are dominated by bacteria that have a high affinity for organic matter (OM) and are composed of specialist species that are always found in association with cyanobacteria and generalist species that can grow in any OM rich environments. Our results also revealed that the associated BC differs structurally and functionally between diazotrophic and non- diazotrophic cyanobacteria. We also specifically investigated the bacteria involved in the nitrogen cycle knowing that this element, with phosphorus, is often limiting for the growth of cyanobacteria. Our results show that several essential steps in this cycle appear to be rather done by the BC that is not under the direct influence of cyanobacteria, which suggests the existence of a decoupling between the MO and nitrogen cycles between associated and non-associated BC.Les écosystèmes aquatiques eutrophes sont souvent perturbés par des proliférations de cyanobactéries potentiellement toxiques. Si de nombreux travaux ont été publiés sur leur écologie et leur toxicité, peu d'entre eux concernent leurs interactions avec les bactéries chimiotrophes qui leur sont associées au sein de la phycosphère, ce qui a motivé cette thèse. Par des travaux réalisés sur des écosystèmes naturels et des approches en laboratoire, nous montrons que les communautés bactériennes (CB) associées aux cyanobactéries ont une structure et une composition différentes de celles qui ne subissent pas leur influence directe. Ces communautés associées sont dominées par des bactéries ayant une forte affinité pour la matière organique (MO) et réunissant des espèces spécialistes toujours retrouvées en association avec des cyanobactéries et des espèces généralistes capables de se développer dans tous les environnements riches en MO. Nos travaux ont aussi révélé que les CB présentent des différences structurelles et fonctionnelles selon le genre de cyanobactéries auquel elles sont associées, et notamment selon leur capacité à fixer ou non l'azote atmosphérique. Enfin, un focus particulier a été porté sur les bactéries impliquées dans le cycle de l'azote sachant que cet élément est avec le phosphore, souvent limitant pour la croissance des cyanobactéries. Nos résultats montrent que plusieurs étapes essentielles de ce cycle semblent être plutôt effectuées par la CB qui n'est pas sous l'influence directe des cyanobactéries ce qui suggère l'existence d'un découplage dans l'exploitation de la MO et dans le cycle de l'azote entre CB associées et non associées aux cyanobactéries

Parasite threats from the ornamental fish trade

The ornamental fish trade is an important commodity sector that involves the capture or farming of fish species for their aesthetic value. Since the 1960s, technological advances have enabled multiple countries to trade numerous ornamental fish species globally. As such, the ornamental fish trade is a pathway for the introduction of exotic fish species and their associated parasites and pathogens into endemic environments, with the potential for detrimental effects on biodiversity, ecosystems, industries, and their dependent local communities. Governments can establish quarantine measures to detect, prevent and mitigate the risks of introducing exotic parasites and pathogens. For example, Australia has established import requirements for ornamental fish species based on risk assessments undertaken by the Australian government Department of Agriculture and Water Resources (DAWR). However, Australian risk assessments largely focus on parasites and pathogens of global significance in food fish production (i.e., salmonids and prawns). As such, established biosecurity requirements for the import of ornamental fish to Australia (DAWR 2018), focus on pathogenic bacteria (e.g., Aeromonas salmonicida (Lehmann and Neumann, 1896)) and viruses (e.g., spring viraemia of carp virus (SVCV)) known to impact aquaculture, while a much broader parasite fauna of ornamental fishes remains to be assessed. The aim of this thesis was to address three specific gaps of knowledge of the ornamental trade. First, I examined limitations in data collation of t ornamental fish imported to Australia (Chapter 2). Second, I examined the diversity of parasite fauna infecting traded marine and freshwater ornamental fish species (Chapter 3 and 4), and; third, I evaluated the validity of cutting-edge molecular methods to detect parasites infecting imported ornamental fishes at border control (Chapters 5 and 6). Accurate data that describes the supply and demand of the global ornamental trade is essential for the development of comprehensive biosecurity protocols to protect endemic ecosystems and natural resources from introduced pathogens and parasites. To quantify the species diversity and volume of ornamental fishes imported to Australia, I examined publicly available data of aquarium fish imports to Australia between 2010-2016, collated and curated by DAWR (Chapter 2). I found that DAWR provides publicly available records of imported ornamental fish species ascribed to categories that offered limited resolution regarding the specific species identity. Taxonomically sound evaluation of Australian aquarium imports would be useful to understand the importance of the Australian aquarium trade in the translocation of potentially hazardous parasites and pathogens, and aid international conservation policies. Following, I surveyed freshwater and marine ornamental fish populations imported from Asia (i.e., Singapore, Malaysia, Thailand and Sri Lanka) to Australia for the presence of protozoan (Chapter 3) and metazoan parasites (Chapter 4). Fish were received following veterinary certification by exporting countries declaring no clinical signs of pests or diseases, and visual inspection by Australian Quarantine Services. Fish necropsies revealed a diverse array of parasite species, including 18 putative types of myxozoans (e.g. Ceratomyxa, Kudoa and Myxobolus spp.), and 14 parasitic monogenean species (e.g. Dactylogyrus, Gyrodactylus, Urocleidoides, and Trianchoratus spp.). One of the major findings was that goldfish, Carassius auratus Linnaeus, 1758, which are the most frequently traded freshwater fish species world-wide, exhibited high parasite diversity (Chapter 3 and 4). Subsequently, I conducted an exhaustive review of the history of the goldfish trade and parasite richness to provide insight into how the international trade of this species may have facilitated parasite co-introduction and co-invasion (Chapter 5). I found that more than 113 parasite species infect goldfish in their native range, of which 26 species were likely co-introduced with the international trade of goldfish (or other cyprinids). These included harmful, generalist parasite species in freshwater aquaculture fishes such as Ichthyophthirius multifiliis Fouquet, 1876, Lernaea cyprinacea Linnaeus, 1758, and Schyzocotyle acheilognathi (Yamaguti, 1934). It is concluded that the goldfish trade likely continues to facilitate the introduction and invasion of exotic parasites on a global scale. It is clear that pre-export health requirements for the importation of ornamental fish species into Australia are not being met (Chapters 3-5), and that cryptic parasites are not detected during visual inspections at border control. Thus, inspection prior to exportation and at border control must account for the highly cryptic nature of parasites and pathogens and consider alternatives to current pre-export conditions and visual inspections at border control. For this reason, I proposed screening fish transport water for the presence of parasite environmental DNA (eDNA) as a detection method for enhanced biosecurity (Chapter 6). I examined water samples from 11 target populations (cyprinids susceptible to Dactylogyrus spp. infections) and seven non-target fish populations (non-cyprinids, not susceptible to Dactylogyrus spp. infections) imported from southeast Asia to Australia for the presence of eDNA from five Dactylogyrus species (Monogenea: Dactylogyridae) using novel species-specific quantitative PCR (qPCR) assays. Dactylogyrus spp. eDNA was detected in all targeted fish populations, showing that eDNA presents a considerable advantage over visual inspections and parasitological necropsies. However, Dactylogyrus spp. eDNA was also detected in water from non-cyprinid fish populations that are not susceptible to and were not infected by Dactylogyrus parasites, highlighting the risk of false positive detections associated with contaminated water sources used to transport ornamental fish species. Environmental DNA screening for parasite DNA offers a highly sensitive and non-invasive detection tool during pre-export monitoring of ornamental species and could aid quarantine officers to triage high-risk ornamental fish exports based on eDNA detection of parasite DNA in the exporting country. Nonetheless, quarantine officers should be vigilant in the limitations posed by contaminated water sources if eDNA screening methods are used at border control. Parasite eDNA detection in water samples from non-cyprinid fish populations in Chapter 5 suggested the possibility of false positive detections by eDNA screening. For this reason, I tested the reliability of eDNA screening methods by qPCR for biosecurity purposes in an experimental system simulating the export process (Chapter 7). Experimentally infected live fish (i.e., the monogenean Neobenedenia girellae (Hargis, 1955) infecting Lates calcarifer (Bloch, 1790)) were used to detect parasite eDNA in water samples, simulating the export process from packaging to delivery over a 48 h period. The consignments included 'infected fish', 'treated fish', and 'contaminated water' (containing dead parasites) delivered by 'exporting companies'. Quantitative PCR tests were inaccurate when detecting eDNA collected from low parasite intensities (mean intensity ± S.D. = 6.80 ± 4.78 parasites/fish). Quantitative PCR tests detected parasite eDNA in 50% of infected fish indicating a high plausibility of false negative detections because of low eDNA concentrations in water samples. Furthermore, parasite eDNA was detected in70% of non-infected fish in contaminated water samples, indicating the possibility of false positive detection of DNA from dead parasites present in the water. Environmental DNA screening methods, while more sensitive than current biosecurity protocols, are limited for accurate and reliable use where differentiation between live parasite infections and dead, non-viable parasites in the water is paramount. This thesis highlights the limitations of the DAWR current data collation framework to accurately examine aquarium fish import data and determined that a large diversity of protozoan and metazoan parasites are not detected at border control. Import conditions for ornamental species are not being met by exporting companies. While eDNA screening methods offer a potential tool for the detection of cryptic pathogens, the limitations of this technique need to be considered for development as a detection tool to demonstrate freedom from parasite infection in the ornamental fish trade