Host Genetic and Environmental Factors Shape the Composition and Function of Gut Microbiota in Populations Living at High Altitude.

The human gut microbiota is affected by genetic and environmental factors. It remains unclear how host genetic and environmental factors affect the composition and function of gut microbiota in populations living at high altitudes. We used a metagenome-wide analysis to investigate the gut microbiota composition in 15 native Tibetans and 12 Hans living on the Tibetan Plateau. The composition of gut microbiota differed significantly between these two groups (P < 0.05). The Planctomycetes was the most abundant phyla both in native Tibetans and in Hans. Furthermore, the most relatively abundant phyla for native Tibetans were Bacteroidetes (15.66%), Firmicutes (11.10%), Proteobacteria (1.32%), Actinobacteria (1.10%), and Tenericutes (0.35%), while the most relatively abundant phyla for Hans were Bacteroidetes (16.28%), Firmicutes (8.41%), Proteobacteria (2.93%), Actinobacteria (0.49%), and Cyanobacteria (0.21%). The abundance of the majority of genera was significantly higher in Tibetans than in Hans (P < 0.01). The number of microbial genes was 4.9 times higher in Tibetans than in Hans. The metabolic pathways and clusters of orthologous groups differed significantly between the two populations (P < 0.05). The abundance of carbohydrate-active enzyme modules and antibiotic resistance genes was significantly lower in Tibetans compared to Hans (P < 0.05). Our results suggest that different genetic factors (race) and environmental factors (diets and consumption of antibiotics) may play important roles in shaping the composition and function of gut microbiota in populations living at high altitudes.

Ecotoxicity assessment of microcystins from freshwater samples using a bioluminescent cyanobacterial bioassay.

The hepatotoxic cyanotoxins microcystins (MCs) are emerging contaminants naturally produced by cyanobacteria. Yet their ecological role remains unsolved, previous research suggests that MCs have allelopathic effects on competing photosynthetic microorganisms, even eliciting toxic effects on other freshwater cyanobacteria. In this context, the bioluminescent recombinant cyanobacterium Anabaena sp. PCC7120 CPB4337 (hereinafter Anabaena) was exposed to extracts of MCs. These were obtained from eight natural samples from freshwater reservoirs that contained MCs with a concentration range of 0.04-11.9 µg MCs L-1. MCs extracts included the three most common MCs variants (MC-LR, MC-RR, MC-YR) in different proportions (MC-LR: 100-0%; MC-RR: 100-0%; MC-YR: 14.2-0%). The Anabaena bioassay based on bioluminescence inhibition has been successfully used to test the toxicity of many emerging contaminants (e.g., pharmaceuticals) but never for cyanotoxins prior to this study. Exposure of Anabaena to MCs extracts induced a decrease in its bioluminescence with effective concentration decreasing bioluminescence by 50% ranging from 0.4 to 50.5 µg MC L-1 in the different samples. Bioluminescence responses suggested an interaction between MCs variants which was analyzed via the Additive Index method (AI), indicating an antagonistic effect (AI < 0) of MC-LR and MC-RR present in the samples. Additionally, MC extracts exposure triggered an increase of intracellular free Ca2+ in Anabaena. In short, this study supports the use of the Anabaena bioassay as a sensitive tool to assess the presence of MCs at environmentally relevant concentrations and opens interesting avenues regarding the interactions between MCs variants and the possible implication of Ca2+ in the mode of action of MCs towards cyanobacteria.

Critical Assessment of Analytical Techniques in the Search for Biomarkers on Mars: A Mummified Microbial Mat from Antarctica as a Best-Case Scenario.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

The contribution of volunteer-based monitoring data to the assessment of harmful phytoplankton blooms in Brazilian urban streams.

Urban streams are vulnerable to a range of impacts, leading to the impairment of ecosystem services. However, studies on phytoplankton growth in tropical lotic systems are still limited. Citizen science approaches use trained volunteers to collect environmental data. We combined data on urban streams collected by volunteers with data obtained by professional scientists to identify potential drivers of phytoplankton community and determine thresholds for Cyanobacteria development. We combined datasets (n=117) on water quality and environmental observations in 64 Brazilian urban streams with paired data on phytoplankton. Sampling activities encompassed dry (July 2013 and July 2015) and warm (February and November 2014) seasons. Volunteers quantified phosphate (PO43-), nitrate (NO3-) and turbidity in each stream using colorimetric and optical methods and recorded environmental conditions in the immediate surroundings of the sites through visual observations. We used non-parametric statistics to identify correlations among nutrients, turbidity and phytoplankton. We also looked for thresholds with respect to high Cyanobacteria abundance (>50,000cells/mL). The streams were characterized by relatively high nutrient concentrations (PO43-: 0.11mg/L; NO3-: 2.6mg/L) and turbidity (49 NTU). Phytoplankton densities reached 189,000cells/mL, mainly potentially toxic Cyanobacteria species. Moderate but significant (p<0.05) correlations were observed between phytoplankton density and turbidity (ρ=0.338, Spearman) and PO43- (ρ=0.292), but not with NO3-. Other important variables (river flow, temperature and light) were not assessed. Volunteers' observations covaried with phytoplankton density (p<0.05, Kruskal-Wallis), positively with increasing number of pollution sources and negatively with presence of vegetation in the riparian zone. Our results indicate that thresholds for PO43- (0.11mg/L) can be used to separate systems with high Cyanobacteria density. The number of pollution sources provided a good indicator of waterbodies with potential cyanobacteria problems. Our findings reinforced the need for nutrient abatement and restoration of local streams and highlighted the benefits of volunteer-based monitoring to support decision-making.

A Multiscale Mapping Assessment of Lake Champlain Cyanobacterial Harmful Algal Blooms.

Lake Champlain has bays undergoing chronic cyanobacterial harmful algal blooms that pose a public health threat. Monitoring and assessment tools need to be developed to support risk decision making and to gain a thorough understanding of bloom scales and intensities. In this research application, Landsat 8 Operational Land Imager (OLI), Rapid Eye, and Proba Compact High Resolution Imaging Spectrometer (CHRIS) images were obtained while a corresponding field campaign collected in situ measurements of water quality. Models including empirical band ratio regressions were applied to map chlorophylla and phycocyanin concentrations; all sensors performed well with R² and root-mean-square error (RMSE) ranging from 0.76 to 0.88 and 0.42 to 1.51, respectively. The outcomes showed spatial patterns across the lake with problematic bays having phycocyanin concentrations >25 µg/L. An alert status metric tuned to the current monitoring protocol was generated using modeled water quality to illustrate how the remote sensing tools can inform a public health monitoring system. Among the sensors utilized in this study, Landsat 8 OLI holds the most promise for providing exposure information across a wide area given the resolutions, systematic observation strategy and free cost.

Preliminary Assessment of Cyanobacteria Diversity and Toxic Potential in Ten Freshwater Lakes in Selangor, Malaysia.

Toxic cyanobacteria blooms are increasing in magnitude and frequency worldwide. However, this issue has not been adequately addressed in Malaysia. Therefore, this study aims to better understand eutrophication levels, cyanobacteria diversity, and microcystin concentrations in ten Malaysian freshwater lakes. The results revealed that most lakes were eutrophic, with total phosphorus and total chlorophyll-a concentrations ranging from 15 to 4270 µg L(-1) and 1.1 to 903.1 µg L(-1), respectively. Cyanobacteria were detected in all lakes, and identified as Microcystis spp., Planktothrix spp., Phormidium spp., Oscillatoria spp., and Lyngbya spp. Microcystis spp. was the most commonly observed and most abundant cyanobacteria recorded. Semi-quantitative microcystin analysis indicated the presence of microcystin in all lakes. These findings illustrate the potential health risk of cyanobacteria in Malaysia freshwater lakes, thus magnifying the importance of cyanobacteria monitoring and management in Malaysian waterways.

Optical classification of algae species with a glass lab-on-a-chip.

The identification of submillimetre phytoplankton is important for monitoring environmental and climate changes, as well as evaluating water for health reasons. Current standard methods for phytoplankton species identification require sample collection and ex situ analysis, an expensive procedure which prevents the rapid identification of phytoplankton outbreaks. To address this, we use a glass-based microchip with a microchannel and waveguide included on a monolithic substrate, and demonstrate its use for identifying phytoplankton species. The microchannel and the specimens inside it are illuminated by laser light from the curved waveguide as algae-laden water is passed through the channel. The intensity distribution of the light collected from the biochip is monitored with an external photodetector. Here, we demonstrate that the characteristics of the photodiode signal from this simple and robust system can provide significant and useful information as to the contents of the channel. Specifically, we show first that the signals are correlated to the size of algae cells. Using a pattern-matching neural network, we demonstrate the successful classification of five algae species with an average 78% positive identification rate. Furthermore, as a proof-of-concept for field-operation, we show that the chip can be used to distinguish between detritus in field-collected water and the toxin-producing cyanobacterium Cyanothece.

Determination of microcystin-LR in drinking water using UPLC tandem mass spectrometry-matrix effects and measurement.

A simple detection method using ultra-performance liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS-MS) coupled with the sample dilution method for determining trace microcystin-LR (MC-LR) in drinking water is presented. The limit of detection (LOD) was 0.04 µg/L and the limit of quantitation (LOQ) was 0.1 µg/L. Water matrix effects of ionic strength, dissolved organic carbon (DOC) and pH were examined. The results indicate that signal detection intensity for MC-LR was significantly suppressed as the ionic strength increased from ultrapure water condition, whereas it increased slightly with solution pH and DOC at low concentrations. However, addition of methanol (MeOH) into the sample was able to counter the signal suppression effects. In this study, dilution of the tap water sample by adding 4% MeOH (v/v) was observed to be adequate to compensate for the signal suppression. The recoveries of the samples fortified with MC-LR (0.2, 1, and 10 µg/L) for three different tap water samples ranged from 84.4% to 112.9%.

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.

Sustainable phosphorus loadings from effective and cost-effective phosphorus management around the Baltic Sea.

Nutrient over-enrichment of the Baltic Sea, accompanied by intensified algal blooms and decreasing water clarity, has aroused widespread concern in the surrounding countries during the last four decades. This work has used a well-tested dynamic mass-balance model to investigate which decrease in total phosphorus loading would be required to meet the environmental goal to restore the trophic state in the Baltic Sea to pre-1960s levels. Furthermore, the extent to which various abatement options may decrease the phosphorus loading in a cost-effective manner has been studied. Upgrading urban sewage treatment in the catchment could, alone or in combination with banning phosphates in detergents, be sufficient to meet the set environmental goal, at an estimated annual basin-wide cost of 0.21-0.43 billion euro. Such a plan would potentially decrease the total phosphorus loading to the Baltic Sea with 6,650-10,200 tons per year.

Diversity and similarity of microbial communities in petroleum crude oils produced in Asia.

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

New Zealand risk management approach for toxic cyanobacteria in drinking water.

Cyanobacterial blooms are common seasonal phenomena occurring worldwide in fresh, estuarine and coastal waters, including those used for drinking-water supplies, recreation and stock watering. In New Zealand, the frequency of blooms and their geographic spread is likely to grow with increasing eutrophication and global climate change. The New Zealand Ministry of Health has recently developed national criteria for assessing and managing the risk of toxic cyanobacteria in drinking-water supplies. This paper investigates a cyanobacterial bloom incident in the summer 2002/03 in the Waikato River and hydro lakes, which are a major drinking-water supply for Hamilton City and many other smaller towns along the river. The procedures invoked by the Hamilton City Council and other authorities to deal with this bloom event are considered in terms of the best practice of the day and compared with the Drinking-Water Standards for New Zealand 2005. The presence of cyanobacteria has significant economic effects because of increases in water supply treatment costs or the need to use an alternative source, and there are also social effects from the disruption of recreational use of water bodies and loss of confidence in the quality of reticulated, treated water supplies. Notional evaluation of economic cost of monitoring regimes and control, based on the Waikato River cyanobacterial bloom incident, is also given. The multi-barrier and process-control risk management approach, reliant on good vertical communication systems between central and local government, is an advanced approach useful for any country that regularly experiences cyanobacterial problems.

Production of antibodies against microcystin-RR for the assessment of purified microcystins and cyanobacterial environmental samples.

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive means to detect these toxins, although cross-reactivity characteristics of different antibodies are variable, and most antibodies have been produced against MC-LR. Here, we have produced the first polyclonal antibodies against the commonly occurring variant, MC-RR, and compared them with MC-LR antibodies for the analysis of purified MCs and cyanobacterial environmental samples. Both antisera cross-reacted with all MCs tested, and with the related cyanobacterial hepatotoxin nodularin-R, but not with non-toxic cyanobacterial peptides. In general, better cross-reactivity characteristics were observed with the MC-RR antisera and limits of quantification were lower for most variants, with all MCs tested and nodularin-R having limits of quantification of 0.31 nM or below. The antisera had different affinities to mixtures containing pooled MC-LR and MC-RR, with MC-LR antisera underestimating total MC concentration when MC-RR represented over 70% of the total MC pool. Both antisera correlated well with HPLC-UV data when incorporated into ELISAs to screen previously characterised environmental samples from Aland, Finland. MC-RR antisera are useful for screening samples containing multiple MCs, and particularly for samples primarily containing MC-RR variants.

Cyanobacterial toxins: risk management for health protection.

This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the risk management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles.

Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates.

Cylindrospermopsis raciborskii, a freshwater cyanobacterium of tropical origin, is not only increasingly found in (sub) tropical water bodies, but also in temperate regions. Since this species may produce potent toxins such as cylindrospermopsin (CYN) and paralytic shellfish poisons, its massive occurrence in water bodies used as drinking water sources or for recreation is of major concern. The proliferation of C. raciborskii in German water bodies has been documented for the past decade. We investigated the occurrence of CYN in field populations and isolates of C. raciborskii from two lakes, and assessed the toxicity of culture isolates using the mouse bioassay, primary rat hepatocytes and human derived cell lines. We show for the first time the occurrence of CYN in German water bodies. None of seven isolates of C. raciborskii contained CYN, however, all isolates were toxic to primary rat hepatocytes, human hepatoblastoma (HEP-G2) and human colon adenocarcinoma (CACO-2) cells. Methanolic extracts were more toxic than aqueous extracts. Three isolates tested in the mouse bioassay were toxic at a concentration of 800 mg kg(-1) showing liver and spleen damage and inflammation of the intestine. These results give strong evidence that the German isolates of C. raciborskii contain currently not identified or unknown toxins.

An assessment of biopotential of three cyanobacterial isolates from Antarctic for carotenoid production.

Specific growth rates and carotenoid contents of three Antarctic and tropical strains of cyanobacteria viz. Anabaena sp., Phormidium sp. and Nostoc sp. were compared in batch and mass cultures to assess bio-potential of Antarctic strains for cost-effective carotenoid production. Antarctic strains though exhibited slightly lower specific growth rates, but contained higher carotenoid contents (per unit dry wt.), than tropical strains. Modification of normal composition of BG-11 culture medium, by altering nitrogen and carbon sources resulted in 25-38% increase in carotenoid content in both types of strains. Mass-culture in indoor and semi-outdoor bio-reactors resulted in 39-113% higher carotenoid content in Antarctic strains, compared to their respective tropical strains. The observations suggest that Antarctic cyanobacteria may have potential as superior strains for maximizing the yield of carotenoids.