Molecular characterization of potential microcystin-producing cyanobacteria in Lake Ontario embayments and nearshore waters

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 microg MC-LReq liter(-1). MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.     

Spatial and temporal diversity of microcystins and microcystin-producing genotypes in Oneida Lake, NY

Oneida Lake is a shallow, eutrophic lake with a well-established cyanobacterial population with reported toxic blooms containing hepatotoxic microcystins (MC). Peak bloom events from the summers of 2002 and 2003 were analyzed to determine the principal cyanobacterial genera containing microcystin synthetase (mcy) genes. Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena and Planktothrix sp. indicated that Microcystis sp. was the dominant mcy genotype. This Microcystis clade was split into two distinct sub-clades. Bloom events contained members of both sub-clades with the higher MC concentrations found when both sub-clades were present in near equal proportions. The proportion of Microcystis containing the mcyD gene ranged from 0 to 37% of the total Microcystis population as determined by quantitative PCR (qPCR). The total concentration of Microcystis containing mcyD genes was linearly related to the concentration of MCs (r² = 0.63). The relationship between mcy genotype and physiochemical variables was examined to determine the factor(s) controlling the periodicity in MC production in Oneida Lake. Multivariate statistical analyses, used to correlate the continuous-response variables, revealed a strong relationship between chlorophyll a, MCs and total Microcystis.     

Phylogenies of Microcystin-Producing Cyanobacteria in the Lower Laurentian Great Lakes Suggest Extensive Genetic Connectivity

Lake St. Clair is the smallest lake in the Laurentian Great Lakes system. MODIS satellite imagery suggests that high algal biomass events have occurred annually along the southern shore during late summer. In this study, we evaluated these events and tested the hypothesis that summer bloom material derived from Lake St. Clair may enter Lake Erie via the Detroit River and represent an overlooked source of potentially toxic Microcystis biomass to the western basin of Lake Erie. We conducted a seasonally and spatially resolved study carried out in the summer of 2013. Our goals were to: 1) track the development of the 2013 summer south-east shore bloom 2) conduct a spatial survey to characterize the extent of toxicity, taxonomic diversity of the total phytoplankton population and the phylogenetic diversity of potential MC-producing cyanobacteria (Microcystis, Planktothrix and Anabaena) during a high biomass event, and 3) compare the strains of potential MC-producers in Lake St. Clair with strains from Lake Erie and Lake Ontario. Our results demonstrated a clear predominance of cyanobacteria during a late August bloom event, primarily dominated by Microcystis, which we traced along the Lake St. Clair coastline downstream to the Detroit River''s outflow at Lake Erie. Microcystin levels exceeded the Province of Ontario Drinking Water Quality Standard (1.5 µg L⁻¹) for safe drinking water at most sites, reaching up to five times this level in some areas. Microcystis was the predominant microcystin producer, and all toxic Microcystis strains found in Lake St. Clair were genetically similar to toxic Microcystis strains found in lakes Erie and Ontario. These findings suggest extensive genetic connectivity among the three systems.     

Microcystis Genotype Succession and Related Environmental Factors in Lake Taihu during Cyanobacterial Blooms

From spring to autumn, heavy Microcystis blooms always occur in Lake Taihu, although environmental conditions vary markedly. We speculated that Microcystis genotype succession could play an important role in adaptation to environmental changes and long-term maintenance of the high Microcystis biomass. In this study, we investigated Microcystis genotype succession pattern and the related environmental variables in Lake Taihu during cyanobacterial blooms. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction -amplified the genus-specific cpcBA and mcyJ gene fragments was used to monitor the variations of Microcystis genotype and potential microcystin (MC)-producing Microcystis genotype compositions during blooms biweekly in three sites (Meiliang Bay, lake center, and Gonghu Bay) and CANOCO 4.5 for Windows were used for the multivariate statistical analysis of their relationships to environmental variables. DGGE patterns indicated that the number of dominant cpcBA genotype per sample increased from spring to autumn. Principal component analysis ordination plots of DGGE profiles showed clear temporal distribution pattern, but not spatial distribution pattern based on both cpcBA and mcyJ genotype compositions. These results indicated there were relatively gradual successions of Microcystis cpcBA and mcyJ genotype compositions in each site, and no distinct spatial difference among the three sites. Redundancy analyses of the gel patterns showed that, in all the three sites, three environmental factors (nitrate, pH, and chemical oxygen demand) were correlated significantly to successions of both cpcBA and mcyJ genotypes except for mcyJ genotype in the lake center. Spearman’s correlations indicated that the three environmental variables were also strongly correlated with chl a and MC concentrations. These results suggested that the environmental factors affecting succession of Microcystis community composition might also influence the growth of Microcystis and MC production.     

Novel Clostridium populations involved in the anaerobic degradation of Microcystis blooms

Understanding the microbial degradation of Microcystis biomass is crucial for determining the ecological consequences of Microcystis blooms in freshwater lakes. The purpose of this study was to identify bacteria involved in the anaerobic degradation of Microcystis blooms. Microcystis scum was anaerobically incubated for 90 days at three temperatures (15 °C, 25 °C and 35 °C). We used terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes, followed by cloning and sequencing of selected samples, to reveal the community composition of bacteria and their dynamics during decomposition. Clostridium spp. were found to be the most dominant bacteria in the incubations, accounting for 72% of the sequenced clones. Eight new clusters or subclusters (designated CLOS.1–8) were identified in the Clostridium phylogenetic tree. The bacterial populations displayed distinct successions during Microcystis decomposition. Temperature had a strong effect on the dynamics of the bacterial populations. At 15 °C, the initial dominance of a 207-bp T-RF (Betaproteobacteria) was largely substituted by a 227-bp T-RF (Clostridium, new cluster CLOS.2) at 30 days. In contrast, at 25 °C and 35 °C, we observed an alternating succession of the 227-bp T-RF and a 231-bp T-RF (Clostridium, new cluster CLOS.1) that occurred more than four times; no one species dominated the flora for the entire experiment. Our study shows that novel Clostridium clusters and their diverse consortiums dominate the bacterial communities during anaerobic degradation of Microcystis, suggesting that these microbes'' function in the degradation process.     

A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean

A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S–23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2–9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.     

Recurring Seasonal Dynamics of Microbial Communities in Stream Habitats

Recurring seasonal patterns of microbial distribution and abundance in three third-order temperate streams within the southeast Pennsylvania Piedmont were observed over 4 years. Populations associated with streambed sediments and rocks (epilithon) were identified using terminal restriction length polymorphism (tRFLP) and sequencing of 16S rRNA genes selectively amplified with primers for the bacterial domain. Analyses of the relative magnitudes of tRFLP peak areas by using nonmetric multidimensional scaling resolved clear seasonal trends in epilithic and sediment populations. Oscillations between two dominant groups of epilithic genotypes, explaining 86% of the seasonal variation in the data set, were correlated with temperature and dissolved organic carbon. Sequences affiliated with epilithic phototrophs (cyanobacteria and diatom chloroplasts), a Rhodoferax sp., and a Bacillus species clustered in the summer, whereas sequences most closely related to “Betaproteobacteria” (putative Burkholderia sp.), and a putative cyanobacterium clustered in the fall/spring. The sediment genotypes also clustered into two groups, and these explained 85% of seasonal variation but correlated only with temperature. A summer tRFLP pattern was characterized by prevalence of “Betaproteobacteria,” “Gammaproteobacteria,” and a Bacillus sp., whereas the winter/spring pattern was characterized by phylotypes most closely related to “Firmicutes,” “Gammaproteobacteria,” and “Nitrospirae.” A close association between these headwater streams and their watersheds was suggested by the recovery of sequences related to microbial populations provisionally attributed to not only freshwaters but also terrestrial habitats.     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.     

Toxic Microcystis is Widespread in Lake Erie: PCR Detection of Toxin Genes and Molecular Characterization of Associated Cyanobacterial Communities

During the past decade, algae blooms, which include the toxic cyanobacterium Microcystis, have reoccurred in the Laurentian Great Lakes, most commonly in the western basin of Lake Erie. Whereas the western basin is the most impacted by toxic Microcystis in Lake Erie, there has historically been little effort focused on identifying the spatial distribution of Microcystis throughout this lake. To address this lack of knowledge, we have employed a polymerase-chain-reaction-based detection of genes required for synthesis of the toxin microcystin (mcyD and mcyB), as well as 16S rDNA fragments specific to either all Microcystis or all cyanobacteria. Using a multiplex approach, we tested 21 samples from 13 field stations and found that toxigenic Microcystis were present in the western and eastern basins in the summers of 1999, 2000, and 2002 and the central basin in 1999 and 2002. This is the most extensive distribution of Microcystis reported in Lake Erie. Clone libraries (16S rDNA) of these cyanobacterial communities were generated from 7 of the 13 field stations (representing all three basins) to partially characterize this microbial community. These libraries were shown to be dominated by sequences assigned to the Synechococcus and Cyanobium phylogenetic cluster, indicating the importance of picoplankton in this large lake system.     

Identification of Freshwater Phycodnaviridae and Their Potential Phytoplankton Hosts, Using DNA pol Sequence Fragments and a Genetic-Distance Analysis

Viruses that infect phytoplankton are an important component of aquatic ecosystems, yet in lakes they remain largely unstudied. In order to investigate viruses (Phycodnaviridae) infecting eukaryotic phytoplankton in lakes and to estimate the number of potential host species, samples were collected from four lakes at the Experimental Lakes Area in Ontario, Canada, during the ice-free period (mid-May to mid-October) of 2004. From each lake, Phycodnaviridae DNA polymerase (pol) gene fragments were amplified using algal-virus-specific primers and separated by denaturing gradient gel electrophoresis; 20 bands were extracted from the gels and sequenced. Phylogenetic analysis indicated that freshwater environmental phycodnavirus sequences belong to distinct phylogenetic groups. An analysis of the genetic distances “within” and “between” monophyletic groups of phycodnavirus isolates indicated that DNA pol sequences that differed by more than 7% at the inferred amino acid level were from viruses that infect different host species. Application of this threshold to phylogenies of environmental sequences indicated that the DNA pol sequences from these lakes came from viruses that infect at least nine different phytoplankton species. A multivariate statistical analysis suggested that potential freshwater hosts included Mallomonas sp., Monoraphidium sp., and Cyclotella sp. This approach should help to unravel the relationships between viruses in the environment and the phytoplankton hosts they infect.     

Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.     

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10⁵ cells ml⁻¹ and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.     

The rise of potentially toxin producing cyanobacteria in Lake Naivasha,Great African Rift Valley,Kenya

Lake Naivasha, an important inland water ecosystem and a crucial freshwater resource in the Great African Rift Valley, has displayed clear signals of degradation in recent decades. We studied the phytoplankton composition and biomass levels in the period 2001–2013 and noted a progressive increase in the occurrence of potentially toxic cyanobacteria. Analyses for the presence of cyanotoxins such as microcystins (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a) were carried out on samples collected in 2008–2013. Among the cyanotoxins tested, low concentrations of MC were detected in the lake. This is the first record of the occurrence of MC in Lake Naivasha. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Lake Naivasha were carried out to establish the taxa of the dominant species. Amplification of the aminotrasferase (AMT) domain responsible for cyanotoxin production confirmed the presence of the mcyE gene belonging to the microcystin synthesis gene cluster in field samples containing Microcystis and Planktothrix species. These findings suggest that toxin producing cyanobacteria could become a threat to users of this over-exploited tropical lake in the near future.     

Competition between the cyanobacterium Microcystis aeruginosa and the diatom Cyclotella sp. under nitrogen-limited condition caused by dilution in eutrophic lake

The improvement of water quality in Lake Tega, Japan, has been carried out by dilution, causing the shift of dominant species from Microcystis aeruginosa to Cyclotella sp. in summer. The disappearance of Microcystis blooms would be related to dilution, but the detail effect has not been understood yet. In this study, the effect of nitrate concentration on the competition between M. aeruginosa and Cyclotella sp. was investigated through the single-species and the competitive culture experiments. The single-species culture experiment indicated that the half saturation constants for M. aeruginosa and Cyclotella sp. were 0.016 and 0.234?mg?N L^?1, representing that M. aeruginosa would possess a higher affinity to nitrate. On the other hand, the maximum growth rate for Cyclotella sp. was obtained as 0.418?day^?1, which did not represent a significant difference with 0.366?day^?1 obtained for M. aeruginosa. The competitive culture experiment revealed that Cyclotella sp. completely dominated over M. aeruginosa at the nitrate concentrations of 0.5 and 2.5?mg?N L^?1. The dominance of Cyclotella sp. could be attributed to the difference in the abilities of nitrate storage as well as nitrate uptake. One of the possibilities for the disappearance of Microcystis blooms caused by dilution as observed in Lake Tega could be due to the decrease in nitrate concentration, and the lower N:P ratio seemed not to relate to Microcystis blooms.     

16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.     

Bacterial Community Composition of Size-Fractioned Aggregates within the Phycosphere of Cyanobacterial Blooms in a Eutrophic Freshwater Lake

Bacterial community composition of different sized aggregates within the Microcystis cyanobacterial phycosphere were determined during summer and fall in Lake Taihu, a eutrophic lake in eastern China. Bloom samples taken in August and September represent healthy bloom biomass, whereas samples from October represent decomposing bloom biomass. To improve our understanding of the complex interior structure in the phycosphere, bloom samples were separated into large (>100 µm), medium (10–100 µm) and small (0.2–10 µm) size aggregates. Species richness and library coverage indicated that pyrosequencing recovered a large bacterial diversity. The community of each size aggregate was highly organized, indicating highly specific conditions within the Microcystis phycosphere. While the communities of medium and small-size aggregates clustered together in August and September samples, large- and medium-size aggregate communities in the October sample were grouped together and distinct from small-size aggregate community. Pronounced changes in the absolute and relative percentages of the dominant genus from the two most important phyla Proteobacteria and Bacteroidetes were observed among the various size aggregates. Bacterial species on large and small-size aggregates likely have the ability to degrade high and low molecular weight compounds, respectively. Thus, there exists a spatial differentiation of bacterial taxa within the phycosphere, possibly operating in sequence and synergy to catalyze the turnover of complex organic matters.     

Boreal Tintinnid Assemblage in the Northwest Pacific and Its Connection with the Japan Sea in Summer 2014

Tintinnids are planktonic ciliates that play an important role in marine ecosystem. According to their distribution in the world oceans, tintinnid genera were divided into several biogeographical types such as boreal, warm water, austral and neritic. Therefore, the oceanic tintinnid assemblage could be correspondingly divided into boreal assemblage, warm water assemblage and austral assemblage. The purpose of this study was to investigate the characteristics of boreal tintinnid assemblage in the Northwest Pacific and the Arctic, and to identify the connection between boreal tintinnid assemblage and neighboring assemblages. Surface water samples were collected along a transect from the East China Sea to the Chukchi Sea in summer 2014. According to the presence of boreal genera and warm water genera, three tintinnid assemblages (the East China Sea neritic assemblage, the Japan Sea warm water assemblage, and the boreal assemblage) were identified along the transect. The boreal assemblage extended from the Chukchi Sea to the waters north of the Sōya Strait. Densities peaks occurred at stations in the two branches of the Alaska Current and decreased both northward and southward. The densities were <10 ind./dm³ at most stations in Arctic region. The dominant genera (Acanthostomella, Codonellopsis, Parafavella, and Ptychocylis) accounted for 79.07±29.67% (n = 49) of the abundance in the boreal assemblage. The densities of the dominant genera covaried with strongly significant positive correlations. Tintinnids with lorica oral diameter of 22–26 μm and 38–42 μm were dominant and contributed 67.35% and 15.13%, respectively, to the total abundance in the boreal assemblage. The distribution and densities of tintinnids in the study area suggest that the Sōya Strait might be a geographical barrier for tintinnids expansion.     

Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake

Cyanobacteria of genus Microcystis sp. have been commonly found in Lake Erie waters during recent summer seasons. In an effort to elucidate relationships between microcystin production, genotypic composition of Microcystis community and environmental parameters in a large lake ecosystem, we collected DNA samples and environmental data during a three-year (2003–2005) survey within Lake Erie and used the data to perform a series of correlation analyses. Cyanobacteria and Microcystis genotypes were quantified using quantitative real-time PCR (qPCR). Our data show that Microcystis in Lake Erie forms up to 42% of all cyanobacteria, and that Microcystis exists as a mixed population of potentially toxic and (primarily) non-toxic genotypes. In the entire lake, the total abundance of Microcystis as well as the abundance of microcystin-producing Microcystis is strongly correlated with the abundance of cyanobacteria suggesting that Microcystis is a significant component of the cyanobacterial community in Lake Erie during summer seasons. The proportion of total Microcystis of all cyanobacteria was strongly linked to the microcystin concentrations, while the percentage of microcystin-producing genotypes within Microcystis population showed no correlation with microcystin concentrations. Correlation analysis indicated that increasing total phosphorus concentrations correlate strongly with increasing microcystin concentrations as well as with the total abundance of Microcystis and microcystin-producing Microcystis.