The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of ...molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.
•Revealed differing fate and temporal dynamics of 4 novel freshwater cyanophages.•Cyanophage were found to be key factors affecting cyanobacteria population dynamic.•Cyanophage infection may shift ...toxin-production of cyanobacteria community.•pH and rainfall as key regulators of both cyanophage and cyanobacteria community.
Considerable studies have been conducted to quantify and examine free cyanophage particles across a wide range of aquatic systems, but few studies have analyzed the dynamics of host cell fraction cyanophages, and even less is known about tropical freshwater cyanophages. Here, we employed qPCR to examine the abundances of four ecologically relevant novel cyanophages (PA-SR01, S-SRP01, S-SRP02 and S-SRM01) in a tropical urban lake in Singapore from July to November 2019. Besides quantifying the extracellular free cyanophage particles, host cell fraction cyanophage were also examined to represent the abundance of intracellular and attached phages. Our study revealed differing fate and dynamics of each cyanophage target. Host cell fraction phage abundance was found to be more abundant than free phage abundance, indicating high level of infection and rapid dispersal of phage particles upon lysis. By further investigating the correlations between free phage particle abundance and numerous biotic (total cyanobacteria, Microcystis abundances, etc) as well as abiotic (nutrients and physiochemical water quality) variables, multiple correlations across different sampling sites were obtained, suggesting pH and rainfall to be potential key factors driving shifts in both cyanophage and cyanobacteria population. This study demonstrated the ecological importance and key roles of the cyanophage PA-SR01, S-SRP01, S-SRP02 and S-SRM01 in tropical freshwater lakes not only as regulators of the cyanobacterial community, but also as promoters of certain cyanotoxin production.
This study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in Singapore and the geospatial distribution of the virome under different ...landuse patterns. Correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. Overall, the majority (65.5%) of the annotated virome belonged to bacteriophages. The percentage of Caudovirales was higher in reservoirs whereas the percentages of Dicistroviridae, Microviridae and Circoviridae were higher in tributaries. Reservoirs showed a higher Shannon-index virome diversity compared to upstream tributaries. Land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. Dicistroviridae and Microviridae were enriched in urbanized tributaries while Mimiviridae, Phycodnaviridae, Siphoviridae and Podoviridae were enriched in parkland reservoirs. Several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. In addition, the relative abundance of PMMoV (pepper mild mottle virus) sequences was significantly correlated with RT-qPCR measurements (0.588 < r < 0.879, p < 0.05). This study shows that spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers of the viral community structure in tropical freshwater ecosystems.
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•Land use is the main driver of the viral community structure in surface waters.•66 viral families were detected and 66% of the annotated virome was bacteriophages.•Reservoirs showed a higher Shannon diversity (10.4) than upstream tributaries (8.4).•Sequences closely related to human picobirnavirus and cyclovirus were detected.•The relative abundance of PMMoV sequences was correlated with RT-qPCR measurements.
In this study, Illumina MiSeq sequencing technique was employed to explore the characteristics and dynamics of cyanobacteria-heterotrophic bacteria between two estuarine reservoirs in sub-tropical ...(reservoir A in Shanghai) and tropical (reservoir B in Singapore) regions. The results indicated that significant differences in bacterial community composition were found between two estuarine reservoirs, which influenced by varied environmental variables. The environmental heterogeneity in reservoir A was much higher, which indicated that the composition of bacterial community in reservoir A was more complex. In contrast, reservoir B provided a suitable and temperate water environment conditions for bacterial growth, which resulted in higher community diversity and less co-exclusion correlations. The molecular ecological network indicated that the presence of dominant bacterial community in each of the reservoir were significant different. These differences mainly reflected the responses of bacterial community to the variations of environmental variables. Although
was the dominant cyanobacterial species in both reservoirs, it exhibited co-occurrence patterns with different heterotrophic bacteria between reservoirs. In addition, the cyanobacteria-heterotrophic bacteria interaction exhibited highly dynamic variations, which was affected by nutrition and survive space. Also, the co-occurrence of
and
found in reservoir B implied that the non-N-fixing
accompanied with N-fixing
occurrence in freshwater lakes, so as to better meet the demand for nitrogen source.
▶
Microcystis spp. was the main toxin producer in Kranji Reservoir. ▶ High temperature, light and nutrient led to high, constant
Microcystis population. ▶ Toxic cell percentage was higher than ...studies in subtropical and temperate regions. ▶
Microcystis population was controlled by the availability of nutrient.
The temporal variations in cyanobacteria and microcystin production in a tropical reservoir, Kranji Reservoir in Singapore, were studied from February 2008 to August 2009 by using molecular techniques and enzyme-linked immunosorbent assay (ELISA) respectively. Results from PCR and qPCR showed that
Microcystis and
Anabaena were present (mean concentrations 4.16
×
10
6
gene
copies/mL and 4.47
×
10
4
gene
copies/mL respectively) and were well correlated to each other (
P
<
0.001) but that
Microcystis was the primary microcystin producer. The average percentage of toxigenic
Microcystis spp. was 55.92%, whereas no
Anabaena-specific microcystin producing gene was detected.
Microcystis-specific microcystin-producing genes
mcyB and
mcyD were detected in all the samples, together with dominant detection of
mcyA and
mcyE genes. However, a low positive detection rate was observed for
mcyC. Due to the combined effects of high temperature, light and nutrient conditions in this tropical reservoir, the
Microcystis cell concentrations were higher and more uniform compared to other
Microcystis blooms reported in subtropical and temperate regions. Furthermore, the proportion of toxigenic
Microcystis in the total
Microcystis population was considerably higher. Principle component analysis and multiple linear regression analysis showed that total nitrogen and total phosphorus were positively correlated with the abundances of total
Microcystis and toxigenic
Microcystis. In addition, total nitrogen, pH and dissolved oxygen were positively correlated with the microcystin concentration.
Microcystin is an algal toxin that is commonly found in eutrophic freshwaters throughout the world. Many studies have been conducted to elucidate the factors affecting its production, but few studies ...have attempted mechanistic models of its production to aid water managers in predicting its occurrence. Here, a mechanistic model was developed based on microcystin production by
spp. under laboratory culture and ambient field conditions. The model was built on STELLA, a dynamic modelling software, and is based on constitutive cell quota that varies with nitrogen, phosphorus, and temperature. In addition to these factors, varying the decay rate of microcystin according to its proportion in the intracellular and extracellular phase was important for the model's performance. With all these effects, the model predicted most of the observations with a model efficiency that was >0.72 and >0.45 for the lab and field conditions respectively. However, some large discrepancies were observed. These may have arisen from the non-constitutive microcystin production that appear to have a precondition of nitrogen abundance. Another reason for the large root mean square error is that cell quota is affected by factors differently between strains.
In this study, we focused on the dynamics of bacterial community composition in a large reservoir in the Yangtze estuary during spring and summer seasons, especially the variations of functional ...mechanisms of microbial community during the seasonal alternation between spring and summer. Both 16S rRNA gene sequencing and shotgun metagenomic sequencing technology were used for these purposes. The results indicated that obvious variations of bacterial community structures were found at different sites. Particle-associated bacterial taxa exhibited higher abundance at the inlet site, which was closer to the Yangtze River with a high level of turbidity. In other sites,
, as the most dominant cyanobacterial species, revealed high abundance driven by increased temperature. Moreover, some heterotrophic bacterial taxa revealed high abundance following the increased
in summer, which indicated potential correlations about carbon source utilization between these microorganisms. In addition, the shotgun metagenomic data indicated during the period of seasonal alternation between spring and summer, the carbohydrate transport and metabolism, energy production and conversion, translation/ribosomal biogenesis, and cell wall/membrane/envelope biogenesis were significantly enhanced at the exit site. However, the course of cell cycle control/division was more active at the internal site.
The increased antibiotic pollutants in aquatic environments pose severe threats on microbial ecology due to their extensive distribution and antibacterial properties. A total of 16 antibiotics ...including fluoroquinolones (FQs) (ofloxacin (OFX), ciprofloxacin (CFX), norfloxacin (NFX)), Sulfonamides (SAs) (sulfamonomethoxine (SMM), sulfadiazine (SDZ), sulfaquinoxaline (SQX)), Tetracyclines (TCs) (tetracycline (TC), doxycycline (DC)), β-lactams (penicillin G (PEN G), penicillin V (PEN V), cefalexin (LEX)), Macrolides (MLs) (erythromycin-H2O (ETM), tylosin (TYL)) and other antibiotics (Polymix-B (POL), Vancomycin (VAN), Lincomycin (LIN)) were detected in the surface water of the Qingcaosha Reservoir. Multivariate statistical analysis indicated that both water quality and physicochemical indexes have less contributions on variations of these antibiotics, suggesting the concentrations of antibiotics inside the reservoir are mainly affected by upstream runoff and anthropic activity along the river. Antibiotics including TYL, PEN G and ETM showed significant correlations with variations of bacterial community composition, and closely connected with various gram-negative bacteria in co-occurrence/exclusion patterns of the network, suggesting these bacterial taxa play important roles in the course of migration and transformation of related antibiotics. In conclusion, further research is required to evaluate the potential risk of genetic transfer of resistance to related bacteria induced by long-term exposure to low levels of antibiotics in the environment.
Rapid proliferation of cyanobacteria in both benthic and suspended (planktonic) habitats is a major threat to environmental safety, as they produce nuisance compounds such as cytotoxins and ...off-flavors, which degrade the safety and quality of water supplies. Temperature and light irradiance are two of the key factors in regulating the occurrence of algal blooms and production of major off-flavors. However, the role of these factors in regulating the growth and metabolism is poorly explored for both benthic and planktonic cyanobacteria. To fill this gap, we studied the effects of light and temperature on the growth and metabolic profiling of both benthic (
sp. MRB220) and planktonic (
sp. SR001) environmental species collected from a freshwater reservoir in Singapore. Moreover, this study is the first report on the metabolic profiling of cyanobacteria belonging to two different habitats in response to altered environmental conditions. The highest growth rate of both species was observed at the highest light intensity (100 μmol photons/m²/s) and at a temperature of 33 °C. Systematic metabolite profiling analysis suggested that temperature had a more profound effect on metabolome of the
, whereas light had a greater effect in the case of
. Interestingly,
sp. SR001 showed a specialized adaptation mechanism via biosynthesis of arginine, and metabolism of cysteine and methionine to survive and withstand higher temperatures of 38 °C and higher. Hence, the mode of strategies for coping with different light and temperature conditions was correlated with the growth and alteration in metabolic activities for physiological and ecological adaptations in both species. In addition, we putatively identified a number of unique metabolites with a broad range of antimicrobial activities in both species in response to both light and temperature. These metabolites could play a role in the dominant behavior of these species in suppressing competition during bloom formation. Overall, this study elucidated novel insights into the effects of environmental factors on the growth, metabolism, and adaptation strategies of cyanobacteria from two different habitats, and could be useful in controlling their harmful effects on human health and environmental concerns.
A non-axenic unialgal culture containing a Subsection V (Stigonematales) cyanobacterium, Hapalosiphon strain MRB 220, was obtained from a benthic freshwater algal mat through multiple transfers ...following growth in sterile media. Physiological characterization demonstrated the culture was capable of nitrogen-fixation and production of the off flavor compound 2-methylisoborneol (2-MIB). Total DNA isolated from this culture was sequenced using Illumina HiSeq and de novo assembled into contigs. The genome of MRB 220 was separated from co-occurring heterotrophic bacteria using sequence homology and compositional approaches, and its purity was confirmed based on best BLAST hit classification and principle component analysis of the tetranucleotide frequencies of fragmented contigs. The genome of ~7.4 Mbp contains 6,345 protein coding genes with 4,320 of these having functional prediction including predicted pathways for biosynthesis of the secondary metabolite welwitindolinone. Analyses of 16S rRNA gene and whole genome sequence average nucleotide identity indicated close relatedness of MRB 220 to the genera Hapalosiphon and Fischerella within the order Stigonematales. Microscopic examination showed that MRB 220 formed heterocystous branched filaments, thereby supporting identification of strain MRB 220 as a morphospecies of Hapalosiphon. Availability of the draft genome of Hapalosiphon strain MRB 220 enables future work to elucidate the pathway and dynamics for biosynthesis of 2-MIB and other secondary metabolites and understand the ecology and physiology of Stigonematales cyanobacteria in tropical freshwaters.
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