Amoebae and Legionella pneumophila in saline environments.

Amoeboid protists that harbor bacterial pathogens are of significant interest as potential reservoirs of disease-causing organisms in the environment, but little is known about them in marine and other saline environments. We enriched amoeba cultures from sediments from four sites in the New England estuarine system of Mt. Hope Bay, Massachusetts and from sediments from six sites in the Great Salt Lake, Utah. Cultures of amoebae were enriched using both minimal- and non-nutrient agar plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested polymerase chain reactions (PCR) and primers specific for the genus. Positive samples were then screened with nested amplification using primers specific for the macrophage infectivity potentiator surface protein (mip) gene from L. pneumophila. Forty-eight percent (185 out of 388) of isolated amoeba cultures were positive for the presence of Legionella species. Legionella pneumophila was detected by PCR in 4% of the amoeba cultures (17 out of 388), and most of these amoebae were growing on marine media. Our results show that amoebae capable of growing in saline environments may harbor not only a diverse collection of Legionella species, but also species potentially pathogenic to humans.

Defining DNA-based operational taxonomic units for microbial-eukaryote ecology.

DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

Changes in microbial community structure in the wake of Hurricanes Katrina and Rita.

Hurricanes have the potential to alter the structures of coastal ecosystems and generate pathogen-laden floodwaters thatthreaten public health. To examine the impact of hurricanes on urban systems, we compared microbial community structures in samples collected after Hurricane Katrina and before and after Hurricane Rita. We extracted environmental DNA and sequenced small-subunit rRNA (SSU rRNA) gene clone libraries to survey microbial communities in floodwater, water, and sediment samples collected from Lake Charles, Lake Pontchartrain, the 17th Street and Industrial Canals in New Orleans, and raw sewage. Correspondence analysis showed that microbial communities associated with sediments formed one cluster while communities associated with lake and Industrial Canal water formed a second. Communities associated with water from the 17th Street Canal and floodwaters collected in New Orleans showed similarity to communities in raw sewage and contained a number of sequences associated with possible pathogenic microbes. This suggests that a distinct microbial community developed in floodwaters following Hurricane Katrina and that microbial community structures as a whole might be sensitive indicators of ecosystem health and serve as "sentinels" of water quality in the environment.

Distinct protistan assemblages characterize the euphotic zone and deep sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf Stream).

Protistan diversity was characterized at three locations in the western North Atlantic (Sargasso Sea and Gulf Stream) by sequencing 18S rRNA genes in samples from euphotic (< or = 125 m) and bathypelagic depths (2500 m). A total of 923 partial-length protistan sequences were analysed, revealing 324 distinct operational taxonomic units (OTUs) determined by an automated OTU-calling program set to 95% sequence similarity. Most OTUs were comprised of only one or two sequences suggesting a large but rare pool of protistan diversity. Many OTUs from both depth strata were associated with recently described novel alveolate and stramenopile lineages while many OTUs from the bathypelagic were affiliated with Acantharea, Polycystinea and Euglenozoa and were not observed in euphotic zone libraries. Protistan assemblages from the euphotic zone and the deep sea were largely composed of distinct OTUs; only 28 of the 324 protistan OTUs were detected in both shallow and deep sea clone libraries. The diversity of protistan assemblages in the deep sea was distinctly lower than the diversity of euphotic zone assemblages. Protistan assemblages from the Gulf Stream were the most diverse for either depth strata. Overall, protistan assemblages from different stations but comparable depths were more similar than the assemblages from different depths at the same station. These data suggest that particular groups of protistan OTUs formed distinct 'shallow' and 'deep-sea' assemblages across widely spaced oceanic locales.

A description of seven Antarctic marine gymnamoebae including a new subspecies, two new species and a new genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp.

Seven marine gymnamoebae were isolated from different environments of seawater, slush (pack ice meltwater), and sediment in the Ross Sea area of Antarctica. All amoebae were isolated and maintained at temperatures below 4 degrees C. Growth, rate of locomotion, and general morphology were observed at an environmentally appropriate temperature (1 degrees C) and at room temperature (approximately 25 degrees C). Molecular (srDNA sequences) and microscopical techniques were used to identify the gymnamoebae and establish their phylogenetic affinities. Three isolates (S-131-2, SL-200, and W4-3) were assigned to a psychrophilic subspecies of Neoparamoeba aestuarina, N. aestuarina antarctica n. subsp., one isolate (S-205) was assigned to a new species of Platyamoeba, P. oblongata n. sp., two isolates (W51C#4 & W51C#5) were also assigned to a new species of Platyamoeba, P. contorta n. sp., and one isolate (S-241) was a novel psychrophilic gymnamoeba Vermistella antarctica n. gen. n. sp. Molecular and morphological results revealed that V. antarctica was not related to any described family of gymnamoebae. Strains S-205, W51C#4, and W51C#5 were capable of locomotion at room temperature, while strains SL-200, S-131-2, W4-3, and S-241 exhibited locomotion only below approximately 10 degrees C. Our results imply that the Antarctic environment is host both to cosmopolitan gymnamoebae that have acquired adaptations for existence at low environmental temperature and to apparently novel psychrophilic amoebae described here for the first time.

Kleptoplasty in an Antarctic dinoflagellate: caught in evolutionary transition?

Photosynthetic dinoflagellates contain a diverse collection of plastid types, a situation believed to have arisen from multiple endosymbiotic events. In addition, a number of heterotrophic (phagotrophic) dinoflagellates possess the ability to acquire chloroplasts temporarily by engulfing algae and retaining their chloroplasts in a functional state. These latter relationships typically last from a few days to weeks, at which point the chloroplasts lose function, are digested and replaced with newly acquired plastids. A novel and abundant dinoflagellate related to the icthyotoxic genera Karenia and Karlodinium was recently discovered by us in the Ross Sea, Antarctica. Sequencing of its plastid small subunit ribosomal gene indicated that it did not share evolutionary history with the plastids of Karenia or Karlodinium, but was closely related to the free-living haptophyte Phaeocystis antarctica, a species that often dominates phytoplankton blooms in the Ross Sea. Chloroplast uptake was observed to occur rapidly (within 2 days), with retention in cultures being long-lived (several months) but not permanent. The dinoflagellate was also incapable of growing indefinitely in continuous darkness with algae as prey. Our findings may indicate an emerging endosymbiotic event yielding a dinoflagellate that is presently neither purely phototrophic nor purely heterotrophic, but occupies a niche juxtaposed between these contrasting nutritional modes.

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis.

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.

Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States.

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.