Metagenomic analysis of a freshwater toxic cyanobacteria bloom

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.     

Identification and culturing of cyanobacteria isolated from freshwater bodies of Sri Lanka for biodiesel production

The present study was carried out to investigate cyanobacteria as a potential source for biodiesel production isolated from fresh water bodies of Sri Lanka. Semi mass culturing and mass culturing were carried out to obtain biomass for extracting total lipids. Fatty acid methyl ester (FAME) or biodiesel was produced from extracted lipid by trans-esterification reaction. FAME component was identified using gas chromatography (GC). Atotal of 74 uni-algal cultures were obtained from Biofuel and Bioenergy laboratory of the National Institute of Fundamental Studies (NIFS), Kandy, Sri Lanka. The total lipid content was recorded highest in Oscillatoria sp. (31.9 ± 2.01% of dry biomass) followed by Synechococcus sp. (30.6 ± 2.87%), Croococcidiopsis sp. (22.7 ± 1.36%), Leptolyngbya sp. (21.15 ± 1.99%), Limnothrixsp. (20.73 ± 3.26%), Calothrix sp. (18.15 ± 4.11%) and Nostoc sp. (15.43 ± 3.89%), Cephalothrixsp. (13.95 ± 4.27%), Cephalothrix Komarekiana (13.8 ± 3.56%) and Westiellopsisprolifica (12.80 ± 1.97%). FAME analysis showed cyanobacteria contain Methyl palmitoleate, Linolelaidic acid methyl ester, Cis-8,11,14-eicosatrienoic acid methyl ester, Cis-10-heptadecanoic acid methyl ester, Methyl myristate, Methyl pentadecanoate, Methyl octanoate, Methyl decanoate, Methyl laurate, Methyl tridecanoate, Methyl palmitoleate, Methyl pentadeconoate, Methyl heptadeconoate, Linolaidic acid methyl ester, Methyl erucate, Methyl myristate, Myristoloeic acid, Methyl palmitate, Cis-9-oleic acid methyl ester, Methyl arachidate and Cis-8,11,14-ecosatrieconoic acid methyl ester. The present study revealed that cyanobacteria isolated from Sri Lanka are potential source for biodiesel industry because of their high fatty acid content. Further studies are required to optimize the mass culture conditions to increase thelipid content from cyanobacterial biomass along with the research in the value addition to the remaining biomass.     

中国香港淡水鱼类的生物多样性与保护

Hong Kong Special Administrative Region,China,has an area of1095km2and is located at the edge of thenorthern tropical zone.The landscape is dominated byhills and ravines withflat landrestrictedtothe lowflood-plains and coastal regions.Inthis hilly domain,the smallrocky streams are typically fast flowing at their uplandsources,and graduallyreduceinspeed asthey wind alonghill cuttings to reach the lowlands.While streams arecommonlyfound,rivers are very limited in number.Thefewlong and meanderi…     

Length-Weight Relationship analysis of four freshwater fish Species from Doyang reservoir at Nagaland,India

The LWRs (length–weight relationships) of four freshwater fish species from medium categorized Doyang reservoir at Wokha District, Nagaland were studied, namely as Garra naganensis Hora, 1921; Devario naganensis Chaudhuri, 1912; Chagunius nicholsi Myers, 1924 and Schistura manipurensis Chaudhuri, 1912. Fish sample were collected in every 15 days interval by cast-nets (1.0 × 1.0 cm mesh size covering the 4.5 m² areas), gill-nets (0.4–5.0 cm mesh size), scoop-nets (mesh size with 0.3 × 0.3 cm to 0.6 × 0.6 cm) and also from the local fishermen in between July 2017 to January 2019. The regression value of slope (b) is fallen into 2.860–3.115 range with significant correlation values (r² ≥ .960).     

Benthic freshwater cyanobacteria as indicators of anthropogenic pressures

Freshwater cyanobacteria are hardly used as biological indicators of anthropogenic pressures, possibly for two main reasons: (a) their response to anthropogenic pressures is often poorly known; (b) reliably identifying cyanobacteria species is a challenge for technicians and researchers. We assessed the usefulness of cyanobacteria species as biological indicators of two human stressors: the high orthophosphate input from urban wastewaters; the high nitrate concentration produced by agricultural land use. We analysed variation in benthic cyanobacterial assemblages at 85 sites in South-Central Spain as a response to eight environmental variables: pH, conductivity, temperature, altitude, nitrate, orthophosphate, irrigation land use and non-irrigation land use.Results revealed that conductivity was the main environmental factor that contributed to differences between assemblages. Orthophosphate was more influential for community composition than nitrate. Changes in species composition related to human pressures suggested that some cyanobacteria species (e.g. Nostoc verrucosum, Phormidium autumnale, Plectonema tomasinianum, Rivularia haematites, Tolypothrix distorta) could be useful tools for the bioindication of anthropogenic pressures; while others species provide more information about natural physicochemical reference conditions (Nostoc caeruleum, Phormidium fonticola). Further research into cyanobacteria and macroalgae assemblages in different impacted scenarios could help improve macrophyte indices.     

A new species of Limnocnida (Limnomedusae,Coelenterata) from a freshwater aquarium in India

We report the occurrence of a freshwater medusa from a laboratory aquarium in Ranchi, Bihar, India. The consistent emergence of these medussae coincided with sunny days after heavy and prolonged rainfall in the months of August to October. The material showed similarity with Limnocnide indica Annandale and L. nepalensis Dumont but differed in many respects, and is given the status of a distinct species, Limnocnida biharensis n.sp.Author to whom all correspondence should be addressed.Author to whom all correspondence should be addressed.     

Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii,and taxonomically diverse freshwater cyanobacteria and green algae

Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.     

Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.     

Conservation of freshwater fish resources of India: new approaches, assessment and challenges

The freshwater resources of India are currently experiencing an alarming decline in fish biodiversity due to several factors and as a result, a sizeable portion of fresh water fishes have been categorized as threatened. This emphasizes an immediate need for initiating research and actions for alternative management techniques to protect these aquatic systems. One such option that has potential to protect freshwater ecosystem from numerous threats is the creation of freshwater aquatic sanctuary (FAS) within protected area network. Though similar conservation practices are well established in the terrestrial and marine ecosystem, however, the work on freshwater systems has been very slow and negligible. In the present communication we conceptualized the need and approach for developing FAS within the protected area network based on our observations in the water bodies of the selected wildlife sanctuaries in Northern India as well as success stories of some other countries. In this study we assessed the fish diversity in the selected protected areas of Northern India. The assessment indicated that these sanctuaries harbor 28.26–31.13% of freshwater fishes, which are threatened in other areas. Apart from Indian Major Carps, Tor putitora, Chitala chitala, Pangasius pangasius, Clupisoma gerua, Ailia coila, Aorichthys aor, Wallago attu, Rhinomugil corsula, Ompok pabda, Ombok pabo etc. were the important species encountered in the protected waters. The various issues related to FAS including objectives, approach, potential tools, implementation and management are discussed towards saving endangered fish germplasm resources. Approaches, tools and modus operandi proposed in this communication could be utilized by other developing countries in the region.     

关于中国淡水生态系统生物多样性监测与管理的探讨

China is one of the countries in the world with therichest species biodiversityinfreshwater ecosystem.How-ever,duetothe rapid economic growthandthe continuingincrease of human disturbances and destructions of aquatichabitats,the biodiversity of freshwater ecosystemsis dras-tically declining.Waterbodies become more and more“deserted”of sensitive species.Water areas are reduced,fragmentized,and changed in their hydrodynamics(i.e.damming),causing changes in sedimentation and otherchanges.Forinstance,the area o...     

The biology of carbonate precipitation by cyanobacteria

Summary In the freshwater areas of the Everglades, Florida, U.S.A., carbonate is precipitated in dense cyanobacterial mats. Precipitation is linked with photosynthesis in the mats in a quantitative relationship. On ground of field observations and experiments a model for precipitation in the filamentous cyanobacteriaScytonema is proposed, which links precipitation to bicarbonate use in photosynthesis and subsequent release of OH⁻ ions. Besides supersaturation of the water with respect to carbonate and photosynthetic bicarbonate use, precipitation requires a suitable sheath structure and composition. The characteristics of the sheath seem to be responsible for a distinct crystal morphology in the two generaScytonema andSchizothrix, as well as for the restriction of calcification to the outer sheath inScytonema. In the immediate vicinity of the trichom precipitation seems to be inhibited. Comparison of this form of calcifying cyanobacteria with calcification in calcareous algae shows many similarities and rises the question of the biological significance of calcification or precipitation. The precipitated carbonate shows equilibrium precipitation in its δ oxygen values, while it is enriched in¹³C relative to the ambient water. This agrees with a model of precipitation in which the carbonate derives from the water immediately surrounding the filament. There the water is depleted in¹²C which is preferably taken up for photosynthesis. No respiratory carbon is involved in precipitation. From measurements of the amount of precipitation in the field and in experiments the annual sedimentation rate is estimated to be 0.024 to 0.24 mm. These values fall within the range of laminae thicknesses in fossil algal laminites.     

Community structure of epilithic and endolithic algae and cyanobacteria on cliffs of the Niagara Escarpment

Abstract. In order to understand how communities of rock-inhabiting micro-organisms are spatially organized, rock samples and environmental variables were collected from five cliff sites in Ontario, Canada. Algae and cyanobacteria were extracted from the epilithic zone (rock surface) and endolithic zone (subsurface) of 180 samples, and the frequency of each taxon (genus or larger) was determined. Multivariate statistical analyses were used to determine (1) the variation in community composition on different spatial scales; (2) differences in community composition between surface and subsurface; and (3) the environmental constraints of community structure. 13 taxa of cyanobacteria and 12 taxa of eukaryotic algae were identified in the endolithic zone, and 17 and 14 in the epilithic zone, respectively. The most frequent taxa were similar in both zones, but MANOVA of species frequencies showed significant differences between endolithic and epilithic communities. DCA and CCA showed that geographic-scale variation in community composition was the dominant trend in the data for both surface and subsurface. Cyanobacteria and eukaryotic algae showed a clear separation on the first ordination axis. When the geographic-scale variation was removed in a partial ordination, the separation of organisms by kingdoms disappeared and epi- and endolithic ordinations were no longer similar. Light then appeared to control the endolithic species composition, while other factors, possibly moisture-related, were suggested for the epilithic community. Despite biomass and productivity that varies mostly at scales of < 1m², we conclude here that species composition and its environmental constraints vary at small as well as larger scales.     

Two approaches to obtaining low,extracellular deoxyribonuclease activity in cultures of heterocyst-forming cyanobacteria

Six out of 158 axenic strains of heterocyst-forming cyanobacteria consistently failed to produce circles of clearing in agar medium containing DNA-methyl green. When tested with [³H]DNA and coliphage DNA, supernatant fluids from cultures of two of these strains [University of Texas Culture Collection (UTEX) strain 2014 and 19-6C-C] showed no detectable deoxyribonuclease activity, and such fluids from another two of the six, and four others, showed low but detectable deoxyribonuclease activity. Covalently closed circular (plasmid) DNA was not detectably degraded by supernatant fluids from UTEX 2014 and 19-6C-C and from four of the other strains. When DNA was incubated with whole cells of certain strains, a sereis of fragments of discrete size was produced, perhaps by cell-bound, periplasmic, restriction endonucleases. Inclusion of one-tenth strength saline sodium citrate (SSC) in an eight-fold dilution of the medium of Allen and Arnon had little effect on growth of Anabaena variabilis American Type Culture Collection (ATCC) strain 29413 yet prevented all but slight degradation of plasmid pBR322 or of DNA.     

Isolation of anaerobic oxalate-degrading bacteria from freshwater lake sediments

Enrichment cultures that anaerobically degraded oxalate were obtained from lake sediment inocula. From these, 5 pure cultures of anaerobic oxalate-degrading bacteria were isolated and partially characterized. The isolates were Gram-negative, non-sporeforming, non-motile, obligate anaerobes. Oxalate was required for growth and was stoichiometrically converted to formate; ¹⁴CO₂ was also recovered when ¹⁴C-oxalate was added. Maximal growth occurred when the oxalate concentration was 50 mM. Acetate stimulated growth in the presence of oxalate, however, ¹⁴C-experiments indicated that acetate was only utilized for cell carbon.The isolates were either spiral-shaped or rod-shaped organisms. The first morphotype grew much more slowly than the second and exhibited 13-fold lower cell yields. These isolates represent a new strain of oxalate-degrading bacteria. The second morphotype was similar to the anaerobic oxalate-degrading bacteria previously found in rumen. This report extends the known habitats in which anaerobic oxalate-degrading organisms have been found to include aquatic sediments.     

Rapid extraction of phycobiliproteins from cultured cyanobacteria samples

Cyanobacteria are a valuable and ubiquitous component of marine picophytoplankton that contribute significantly to total carbon biomass and primary productivity of the oceans. They contain water soluble, natively highly fluorescent proteins, phycobiliproteins, that can be considered ideal marker pigments for understanding the distribution and trophic dynamics of picoplankton populations. However, there is no standard protocol for extracting and quantitating these proteins from cyanobacterial cells. Ideally, the cells would be disrupted quickly and efficiently with complete extraction and recovery of the released proteins. For that purpose, we describe a method for extracting phycobiliproteins from a Synechococcus CCMP 833 cyanobacteria culture that utilizes 3% 3-[(3-cholamidopropyl)dimethyammonio]propanesulfonic acid (Chaps) 0.3% asolectin combined with nitrogen cavitation. Extraction efficiencies of greater than 85% were achieved by this method, which requires less than 3h. The analysis of the extracted samples was carried out by capillary electrophoresis with laser-induced fluorescence detection.     

Fecundity of the freshwater catfishes mystus seenghala (sykes), mystus cavasius (ham), wallagonia attu (bloch) and heteropneustes fossilis (bloch) from the plains of Northern India

Fecundity of four species of catfishes, namely, Mystus seenghala (Sykes), Mystus cavasius (Ham), Wallagonia attu (Bloch) and Heteropneustes fossilis (Bloch), was studied. The fecundity of Mystus seenghala and Mystus cavasius varied between 20064 to 46443 and 3314 to 63135 respectively. In Wallagonia attu and Heteropneustes fossilis it varied between 66070 to 453148 and 1375 to 46737 respectively. In all the four species fecundity has a linear relationship with body weight and gonad weight, and a parabolic relationship with length in all the fishes excepting Mystus seenghala, where it is linear. By subjecting the data to multiple regression analysis, it is observed that the fecundity of each species can be most accurately determined by taking two variables together. In all the fishes studied the relative fecundity (Egg/body wt.) does not change significantly either with length or body weight.     

中国淡水三角涡虫染色体组型的研究

采用压片法对不同产地的淡水三角涡虫染色体进行研究,结果表明,湖北省武汉市珞珈山的淡水三角涡虫体细胞中有16条染色体,为二倍体（2n=2x=16m）;江苏省太湖东山岛的淡水三角涡虫体细胞中有24条染色体,为三倍体（2n=3x=24m）;贵州省遵义市凤凰山的淡水三角涡虫体细胞同时存在24条染色体和16条染色体两种分裂相,为混倍体。本文根据组型分析的结果对上述三个产地淡水三角涡虫的分类和染色体倍性进行了初步研究。另外,本研究中还发现涡虫染色体倍性的变化与涡虫生殖方式的转变以及环境温度有一定的联系。     

Isolation and characterization of plasma membranes from cyanobacteria

The two-phase partition system in comparison to the traditional methods used thus far (density gradients) for the isolation of the plasma membrane from cyanobacteria is described. The advantages of the two-phase system are: A short-time preparation of 3–4 h compared to 16–48 h required for the density gradient method; a purer fraction, resulting from separation according to membrane surface charge and hydrophobicity, not specific density; and, ease of scaling-up for obtaining large quantities.
Also, the different biological activities attributed to this membrane to date are summarized. Findings on the typical plasma membrane ATPase (P-type ATPase) as well as the nutrient transporters and the corresponding proteins are included. As for the electron transport chain, one may conclude that this membrane contains a complete system (similar to that of the mitochondrion), portraying apparently F-type (F₀F₁) ATPase activity.     

Growth of an acid-tolerant stonefly on epilithic biofilms from streams of contrasting pH

1. The apparent absence of a specialist herbivorous grazer guild from many acid streams suggests that algae-grazer linkages in acid-stream food webs are weak or absent. It has been hypothesized that the absence of herbivores is a consequence of the low quality and/or quantity of biofilms in acid streams.
2. We compared the taxonomic composition, biomass and potential nutritional quality of epilithic biofilms from four acid and four circumneutral streams, and examined whether nymphs of a herbivore–detritivore, the stonefly Nemurella pictetii (Plecoptera: Nemouridae), could grow equally well when fed on eight biofilms from four acid and four circumneutral streams.
3. Biofilms from acid and circumneutral streams differed strongly in algal composition, the former having relatively more coccoid green algae but fewer diatoms and filamentous green algae. Diatom floras differed with stream water pH.
4. The quantity (i.e. area-specific chlorophyll content, algal numbers and AFDM) and quality (biomass-specific protein and soluble carbohydrate content) of biofilms differed significantly, both among sites of similar pH, and overall between the groups of acid and circumneutral streams.
5. Nymphs of N. pictetii grew successfully on biofilms for 8–10 weeks up to emergence. However, no systematic differences in growth rate were found between the two groups of acid and circumneutral streams. Differences in the digestibility of benthic algae from different sources, and the adjustment of nymphal feeding rates, are discussed in the light of a lack of a clear relationship between growth and food quality.     

Soluble reactive phosphorus (SRP) transport and retention in tropical, rain forest streams draining a volcanic landscape in Costa Rica: in situ SRP amendment to streams and laboratory studies

Soluble reactive phosphorus (SRP) transport/retention was determined in two rain forest streams (Salto, Pantano) draining La Selva Biological Station, Costa Rica. There, SRP levels can be naturally high due to groundwater enriched by geothermal activity within the surfically dormant volcanic landscape, and subsequently discharged at ambient temperature. Combined field and laboratory approaches simulated high but natural geothermal SRP input with the objective of estimating the magnitude of amended SRP retention within high and low SRP settings and determining the underlying mechanisms of SRP retention. First, we examined short-term SRP retention/transport using combined SRP-conservative tracer additions at high natural in situ concentrations. Second, we attempted to observe a DIN response during SRP amendment as an indicator of biological uptake. Third, we determined SRP release/retention using laboratory sediment assays under control and biologically inhibited conditions. Short-term in situ tracer-SRP additions indicated retention in both naturally high and low SRP reaches. Retention of added SRP mass in Upper Salto (low SRP) was 17% (7.5 mg-P m⁻² h⁻¹), and 20% (10.9 mg-P m⁻² h⁻¹) in Lower Salto (high SRP). No DIN response in either nitrate or ammonium was observed. Laboratory assays using fresh Lower Salto sediments indicated SRP release (15.4 ± 5.9 μg-P g dry wt.⁻¹ h⁻¹), when incubated in filter sterilized Salto water at ambient P concentration, but retention when incubated in filter sterilized river water amended to 2.0 mg SRP l⁻¹ (233.2 ± 5.8 μg-P g dry wt.⁻¹ h⁻¹). SRP uptake/release was similar in both control- and biocide-treated sediments indicating predominantly abiotic retention. High SRP retention even under biologically saturated conditions, absence of a DIN response to amendment, patterns of desorption following amendment, and similar patterns of retention and release under control and biologically inhibited conditions all indicated predominantly abiotic P flux.