Temperature dependence of parasitoid infection and abundance of a diatom revealed by automated imaging and classification.

Diatoms are a group of phytoplankton that contribute disproportionately to global primary production. Traditional paradigms that suggest diatoms are consumed primarily by larger zooplankton are challenged by sporadic parasitic "epidemics" within diatom populations. However, our understanding of diatom parasitism is limited by difficulties in quantifying these interactions. Here, we observe the dynamics of Cryothecomonas aestivalis (a protist) infection of an important diatom on the Northeast U.S. Shelf (NES), Guinardia delicatula, with a combination of automated imaging-in-flow cytometry and a convolutional neural network image classifier. Application of the classifier to >1 billion images from a nearshore time series and >20 survey cruises across the broader NES reveals the spatiotemporal gradients and temperature dependence of G. delicatula abundance and infection dynamics. Suppression of parasitoid infection at temperatures <4 °C drives annual cycles in both G. delicatula infection and abundance, with an annual maximum in infection observed in the fall-winter preceding an annual maximum in host abundance in the winter-spring. This annual cycle likely varies spatially across the NES in response to variable annual cycles in water temperature. We show that infection remains suppressed for ~2 mo following cold periods, possibly due to temperature-induced local extinctions of the C. aestivalis strain(s) that infect G. delicatula. These findings have implications for predicting impacts of a warming NES surface ocean on G. delicatula abundance and infection dynamics and demonstrate the potential of automated plankton imaging and classification to quantify phytoplankton parasitism in nature across unprecedented spatiotemporal scales.

Parasitic infections by Group II Syndiniales target selected dinoflagellate host populations within diverse protist assemblages in a model coastal pond.

Protists are integral to marine food webs and biogeochemical cycles; however, there is a paucity of data describing specific ecological niches for some of the most abundant taxa in marker gene libraries. Syndiniales are one such group, often representing the majority of sequence reads recovered from picoplankton samples across the global ocean. However, the prevalence and impacts of syndinian parasitism in marine environments remain unclear. We began to address these critical knowledge gaps by generating a high-resolution time series (March-October 2018) in a productive coastal pond. Seasonal shifts in protist populations, including parasitic Syndiniales, were documented during periods of higher primary productivity and increased summer temperature-driven stratification. Elevated concentrations of infected hosts and free-living parasite spores occurred at nearly monthly intervals in July, August, and September. We suggest intensifying stratification during this period correlated with the increased prevalence of dinoflagellates that were parasitized by Group II Syndiniales. Infections in some protist populations were comparable to previously reported large single-taxon dinoflagellate blooms. Infection dynamics in Salt Pond demonstrated the propagation of syndinian parasites through mixed protist assemblages and highlighted patterns of host/parasite interactions that better reflect many other marine environments where single taxon blooms are uncommon.

Rediscovery of the gonyaulacoid dinoflagellate Schuettiella.

The Gonyaulacales includes some of the most intensely investigated genera of harmful dinoflagellates. The knowledge of the evolutionary relationships is necessary, but incomplete because genera such as Schuettiella have not been studied by contemporary methods. The morphology and molecular phylogeny of type species of the genus Schuettiella, S. mitra, have been investigated from Brazil. The first scanning electron micrographs reveal a distinctive thecal ornamentation with sunken stripes and rows with exclamation mark-shaped pores in the postcingular plates. The Kofoidean plate formula Po, 2', 1a, 6″ is re-interpreted as Po, 3', 1a, 5″, after considering the narrow mid-ventral plate as homologous to the first apical plate, although it does not reach the apex. In the rRNA gene phylogenies, the sequences of S. mitra clustered as an independent lineage closely related to the globular and planktonic Protoceratiaceae (Ceratocorys, Pentaplacodinium, Protoceratium) and the laterally compressed benthic genus Carinadinium (formerly Thecadinium). Schuettiella and Carinadinium are considered members of the Protoceratiaceae. The possession of a single anterior intercalary plate is an apomorphic trait of this family. This reinforces the value of the number of intercalary plates as a diagnostic character for the classification of the gonyaulacalean families despite the differences in the general appearance.

A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis.

Plastid endosymbiosis has been a major force in the evolution of eukaryotic cellular complexity, but how endosymbionts are integrated is still poorly understood at a mechanistic level. Dinoflagellates, an ecologically important protist lineage, represent a unique model to study this process because dinoflagellate plastids have repeatedly been reduced, lost, and replaced by new plastids, leading to a spectrum of ages and integration levels. Here we describe deep-transcriptomic analyses of the Antarctic Ross Sea dinoflagellate (RSD), which harbors long-term but temporary kleptoplasts stolen from haptophyte prey, and is closely related to dinoflagellates with fully integrated plastids derived from different haptophytes. In some members of this lineage, called the Kareniaceae, their tertiary haptophyte plastids have crossed a tipping point to stable integration, but RSD has not, and may therefore reveal the order of events leading up to endosymbiotic integration. We show that RSD has retained its ancestral secondary plastid and has partitioned functions between this plastid and the kleptoplast. It has also obtained genes for kleptoplast-targeted proteins via horizontal gene transfer (HGT) that are not derived from the kleptoplast lineage. Importantly, many of these HGTs are also found in the related species with fully integrated plastids, which provides direct evidence that genetic integration preceded organelle fixation. Finally, we find that expression of kleptoplast-targeted genes is unaffected by environmental parameters, unlike prey-encoded homologs, suggesting that kleptoplast-targeted HGTs have adapted to posttranscriptional regulation mechanisms of the host.

Molecular phylogeny and synonymy of Balechina gracilis comb. nov. (=Gymnodinium gracile), a widespread polymorphic unarmored dinoflagellate (Dinophyceae).

Gymnodinium gracile, described from the coasts of Denmark in 1881, is one of the first described unarmored dinoflagellates. Individuals that morphologically fit with the original description were isolated from the English Channel (North-East Atlantic). The SSU rRNA gene sequences were identical to the sequences identified as Balechina pachydermata and Gymnodinium amphora from the Mediterranean Sea and Brazil. We propose the transfer of Gymnodinium gracile into the genus Balechina as B. gracilis comb. nov. These sequences constitute an independent lineage, clustering with numerous environmental sequences from polar to tropical waters. The widespread distribution, the high plasticity in size, shape and coloration and the difficulties in discerning the fine longitudinal striae have contributed to the description of numerous synonyms: Amphidinium vasculum, Balechina pachydermata (=Gymnodinium pachydermatum), Gymnodinium achromaticum, G. abbreviatum, G. amphora, G. dogielii, G. lohmannii (=G. roseum sensu Lohmann 1908), G. situla, and Gyrodinium cuneatum (=G. gracile sensu Pouchet 1885).

Dinoflagellates Amyloodinium and Ichthyodinium (Dinophyceae), parasites of marine fishes in the South Atlantic Ocean.

The morphology and molecular phylogeny of the parasitic dinoflagellates Ichthyodinium chabelardi and Amyloodinium ocellatum was investigated off Brazil (South Atlantic Ocean). This is the first record of Ichthyodinium and the first molecular data of both parasites from the southern hemisphere. I. chabelardi infected the yolk of eggs of wild populations of Argentine anchovy Engraulis anchoita (Engraulidae) and Brazilian sardinella Sardinella brasiliensis (Clupeidae) in different seasons. The small subunit (SSU) rRNA and internal transcribed spacer (ITS) gene sequences were identical and confirmed Ichthyodinium as a host generalist. The new sequences clustered with the type species I. chabelardi from the North Atlantic and environmental sequences from the Pacific Ocean. A second species from the western Pacific remains undescribed. A. ocellatum was isolated from the gills of a cultured cobia Rachycentron canadum after causing mortality. The SSU rRNA gene sequence of the Brazilian isolate was almost identical to those from the northern hemisphere. This suggests a single species with a widespread distribution, although it is uncertain whether the species has a natural pantropical distribution or is the result of artificial distribution due to human-induced fish transport.

The extraordinary longevity of kleptoplasts derived from the Ross Sea haptophyte Phaeocystis antarctica within dinoflagellate host cells relates to the diminished role of the oxygen-evolving Photosystem II and to supplementary light harvesting by mycosporine-like amino acid/s.

The haptophyte Phaeocystis antarctica and the novel Ross Sea dinoflagellate that hosts kleptoplasts derived from P. antarctica (RSD; R.J. Gast et al., 2006, J. Phycol. 42 233-242) were compared for photosynthetic light harvesting and for oxygen evolution activity. Both chloroplasts and kleptoplasts emit chlorophyll a (Chl a) fluorescence peaking at 683nm (F683) at 277K and at 689 (F689) at 77K. Second derivative analysis of the F689 band at 77K revealed two individual contributions centered at 683nm (Fi-683) and at 689 (Fi-689). Using the p-nitrothiophenol (p-NTP) treatment of Kobayashi et al. (Biochim. Biophys. Acta 423 (1976) 80-90) to differentiate between Photosystem (PS) II and I fluorescence emissions, we could identify PS II as the origin of Fi-683 and PS I as the origin of Fi-689. Both emissions could be excited not only by Chl a-selective light (436nm) but also by mycosporine-like amino acids (MAAs)-selective light (345nm). This suggests that a fraction of MAAs must be proximal to Chls a and, therefore, located within the plastids. On the basis of second derivative fluorescence spectra at 77K, of p-NTP resolved fluorescence spectra, as well as of PSII-driven oxygen evolution activities, PS II appears substantially less active (~1/5) in dinoflagellate kleptoplasts than in P. antarctica chloroplasts. We suggest that a diminished role of PS II, a known source of reactive oxygen species, and a diminished dependence on nucleus-encoded light-harvesting proteins, due to supplementary light-harvesting by MAAs, may account for the extraordinary longevity of RSD kleptoplasts.

Evidence of concurrent local adaptation and high phenotypic plasticity in a polar microeukaryote.

Here we investigated whether there is evidence of local adaptation in strains of an ancestrally marine dinoflagellate to the lacustrine environment they now inhabit (optimal genotypes) and/or if they have evolved phenotypic plasticity (a range of phenotypes). Eleven strains of Polarella glacialis were isolated and cultured from three different environments: the polar seas, a hyposaline and a hypersaline Antarctic lake. Local adaptation was tested by comparing growth rates of lacustrine and marine strains at their own and reciprocal site conditions. To determine phenotypic plasticity, we measured the reaction norm for salinity. We found evidence of both, limited local adaptation and higher phenotypic plasticity in lacustrine strains when compared with marine ancestors. At extreme high salinities, local lake strains outperformed other strains, and at extreme low salinities, strains from the hyposaline lake outperformed all other strains. The data suggest that lake populations may have evolved higher phenotypic plasticity in the lake habitats compared with the sea, presumably due to the high temporal variability in salinity in the lacustrine systems. Moreover, the interval of salinity tolerance differed between strains from the hyposaline and hypersaline lakes, indicating local adaptation promoted by different salinity.

Estimating Protistan Diversity Using High-Throughput Sequencing.

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full-length sequences. A total of 7,432 full-length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full-length 18S rRNA gene sequences. Present high-throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.

Physiological responses of three species of Antarctic mixotrophic phytoflagellates to changes in light and dissolved nutrients.

Antarctic phototrophs are challenged by extreme temperatures, ice cover, nutrient limitation, and prolonged periods of darkness. Yet this environment may also provide niche opportunities for phytoplankton utilizing alternative nutritional modes. Mixotrophy, the combination of photosynthesis and particle ingestion, has been proposed as a mechanism for some phytoplankton to contend with the adverse conditions of the Antarctic. We conducted feeding experiments using fluorescent bacteria-sized tracers to compare the effects of light and nutrients on bacterivory rates in three Antarctic marine photosynthetic nanoflagellates representing two evolutionary lineages: Cryptophyceae (Geminigera cryophila) and Prasinophyceae (Pyramimonas tychotreta and Mantoniella antarctica). Only G. cryophila had previously been identified as mixotrophic. We also measured photoautotrophic abilities over a range of light intensities (P vs. I) and used dark survival experiments to assess cell population dynamics in the absence of light. Feeding behavior in these three nanoflagellates was affected by either light, nutrient levels, or a combination of both factors in a species-specific manner that was not conserved by evolutionary lineage. The different responses to environmental factors by these mixotrophs supported the idea of tradeoffs in the use of phagotrophy and phototrophy for growth.

Contribution of sand-associated enterococci to dry weather water quality.

Culturable enterococci and a suite of environmental variables were collected during a predominantly dry summer at a beach impacted by nonpoint source pollution. These data were used to evaluate sands as a source of enterococci to nearshore waters, and to assess the relationship between environmental factors and dry-weather enterococci abundance. Best-fit multiple linear regressions used environmental variables to explain more than half of the observed variation in enterococci in water and dry sands. Notably, during dry weather the abundance of enterococci in dry sands at the mean high-tide line was significantly positively related to sand moisture content (ranging from <1-4%), and the daily mean ENT in water could be predicted by a linear regression with turbidity alone. Temperature was also positively correlated with ENT abundance in this study, which may indicate an important role of seasonal warming in temperate regions. Inundation by spring tides was the primary rewetting mechanism that sustained culturable enterococci populations in high-tide sands. Tidal forcing modulated the abundance of enterococci in the water, as both turbidity and enterococci were elevated during ebb and flood tides. The probability of samples violating the single-sample maximum was significantly greater when collected during periods with increased tidal range: spring ebb and flood tides. Tidal forcing also affected groundwater mixing zones, mobilizing enterococci from sand to water. These data show that routine monitoring programs using discrete enterococci measurements may be biased by tides and other environmental factors, providing a flawed basis for beach closure decisions.

Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18S rRNA genes.

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

Selective feeding and foreign plastid retention in an Antarctic dinoflagellate.

The peridinin-containing plastid found in most photosynthetic dinoflagellates is thought to have been replaced in a few lineages by plastids of chlorophyte, diatom, or haptophyte origin. Other distinct lineages of phagotrophic dinoflagellates retain functional plastids obtained from algal prey for different durations and with varying source species specificity. 18S rRNA gene sequence analyses have placed a novel gymnodinoid dinoflagellate isolated from the Ross Sea (RSD) in the Kareniaceae, a family of dinoflagellates with permanent plastids of haptophyte origin. In contrast to other species in this family, the RSD contains kleptoplastids sequestered from its prey, Phaeocystis antarctica. Culture experiments were employed to determine whether the RSD fed selectively on P. antarctica when offered in combination with another polar haptophyte or cryptophyte species, and whether the RSD, isolated from its prey and starved, would take up plastids from P. antarctica or from other polar haptophyte or cryptophyte species. Evidence was obtained for selective feeding on P. antarctica, plastid uptake from P. antarctica, and increased RSD growth in the presence of P. antarctica. The presence of a peduncle-like structure in the RSD suggests that kleptoplasts are obtained by myzocytosis. RSD cells incubated without P. antarctica were capable of survival for at least 29.5 months. This remarkable longevity of the RSD's kleptoplasts and its species specificity for prey and plastid source is consistent with its prolonged co-evolution with P. antarctica. It may also reflect the presence of a plastid protein import mechanism and genes transferred to the dinokaryon from a lost permanent haptophyte plastid.

Temporal and spatial variability of constitutive mixotroph abundance and proportion.

Mixotrophic plankton can comprise a substantial portion of the plankton community compared to phytoplankton and zooplankton. However, there is a gap in the understanding of conditions that influence mixotroph prevalence and activity in situ because current methods often over- or underestimate mixotroph abundance. A labeled prey-tracer method was utilized to identify active mixotrophs present at two locations in a temperate estuary over a year. The tracer method was combined with light microscopy data to estimate active mixotroph abundance and proportion. This study estimated that actively grazing mixotrophic taxa were more abundant in the spring and autumn compared to summer. Dinoflagellates typically dominated the mixotrophic taxa except during autumn at the low salinity location when cryptophytes dominated. Further analysis suggested that active mixotroph abundances might not be only regulated by environmental conditions favorable to mixotrophy but, instead, environmental conditions favorable to different mixotrophs utilization of phagotrophy. By focusing on mixotrophic taxa that were identified to be actively grazing at time of sampling, this study provided a more nuanced estimation of mixotroph abundance, increasing the understanding of how mixotrophic abundance and proportion in situ are influenced by the planktonic community composition and environmental factors.

Mixoplankton and mixotrophy: future research priorities.

Phago-mixotrophy, the combination of photoautotrophy and phagotrophy in mixoplankton, organisms that can combine both trophic strategies, have gained increasing attention over the past decade. It is now recognized that a substantial number of protistan plankton species engage in phago-mixotrophy to obtain nutrients for growth and reproduction under a range of environmental conditions. Unfortunately, our current understanding of mixoplankton in aquatic systems significantly lags behind our understanding of zooplankton and phytoplankton, limiting our ability to fully comprehend the role of mixoplankton (and phago-mixotrophy) in the plankton food web and biogeochemical cycling. Here, we put forward five research directions that we believe will lead to major advancement in the field: (i) evolution: understanding mixotrophy in the context of the evolutionary transition from phagotrophy to photoautotrophy; (ii) traits and trade-offs: identifying the key traits and trade-offs constraining mixotrophic metabolisms; (iii) biogeography: large-scale patterns of mixoplankton distribution; (iv) biogeochemistry and trophic transfer: understanding mixoplankton as conduits of nutrients and energy; and (v) in situ methods: improving the identification of in situ mixoplankton and their phago-mixotrophic activity.

Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant Campylobacter jejuni.

We measured ingestion and digestion rates of the pathogenic bacterium Campylobacter jejuni by a freshwater ciliate Colpoda sp. to determine whether Campylobacter is able to resist protist digestion. Campylobacter and the nonpathogenic bacterium Pseudomonas putida LH1 were labeled with a 5-chloromethylfluorescein diacetate, which fluoresces in intact and active cells but fades when exposed to low pH environments, such as protistan food vacuoles. Ingestion and digestion rates were measured via flow cytometry as the change in ciliate fluorescence over time, which corresponded to the quantity of intracellular bacteria. The rate of Campylobacter ingestion exceeded the digestion rate. Ciliates retained labeled Campylobacter 5 h after ingestion was stopped. In contrast, ciliates grazing upon P. putida returned to baseline fluorescence within 5 h, indicating that P. putida were completely digested. The ability of intracellular Campylobacter to remain viable after ingestion was tested by sorting individual ciliates and bacterial cells into Campylobacter-selective media. Campylobacter growth occurred in 15% (± 5 SE) of wells seeded with highly fluorescent ciliates, whereas only 4% (± 1) of wells seeded with free-living Campylobacter exhibited growth. A key advantage of this approach is that it is rapid and should be applicable to other phagocytotis studies.

Diversity of Microbial Eukaryotes Along the West Antarctic Peninsula in Austral Spring.

During a cruise from October to November 2019, along the West Antarctic Peninsula, between 64.32 and 68.37°S, we assessed the diversity and composition of the active microbial eukaryotic community within three size fractions: micro- (> 20 µm), nano- (20-5 µm), and pico-size fractions (5-0.2 µm). The communities and the environmental parameters displayed latitudinal gradients, and we observed a strong similarity in the microbial eukaryotic communities as well as the environmental parameters between the sub-surface and the deep chlorophyll maximum (DCM) depths. Chlorophyll concentrations were low, and the mixed layer was shallow for most of the 17 stations sampled. The richness of the microplankton was higher in Marguerite Bay (our southernmost stations), compared to more northern stations, while the diversity for the nano- and pico-plankton was relatively stable across latitude. The microplankton communities were dominated by autotrophs, mostly diatoms, while mixotrophs (phototrophs-consuming bacteria and kleptoplastidic ciliates, mostly alveolates, and cryptophytes) were the most abundant and active members of the nano- and picoplankton communities. While phototrophy was the dominant trophic mode, heterotrophy (mixotrophy, phagotrophy, and parasitism) tended to increase southward. The samples from Marguerite Bay showed a distinct community with a high diversity of nanoplankton predators, including spirotrich ciliates, and dinoflagellates, while cryptophytes were observed elsewhere. Some lineages were significantly related-either positively or negatively-to ice coverage (e.g., positive for Pelagophyceae, negative for Spirotrichea) and temperature (e.g., positive for Cryptophyceae, negative for Spirotrichea). This suggests that climate changes will have a strong impact on the microbial eukaryotic community.

Rapid shifts in the structure and composition of a protistan assemblage during bottle incubations affect estimates of total protistan species richness.

Changes in the structure and composition of a protistan community were characterized through the analysis of small-subunit ribosomal RNA gene (18S) sequences for a 3-day bottle incubation using a single sample collected in the western North Atlantic. Cloning and sequencing was used to investigate changes in perceived species richness and diversity as a consequence of environmental perturbation. The treatments included a control (unamended seawater), inorganic nutrient enrichment, and enrichment with a complex organic mixture. Five clone libraries were constructed and analyzed at the time of collection (t-0 h) and after 24 (t-24 h) and 72 (t-72 h) h for the control, and at t-72 h for the inorganic and organic enrichments, resulting in an analysis of 1,626 partial 18S rDNA sequences that clustered into 238 operational taxonomic units (OTUs). Analysis of the clone libraries revealed that protistan assemblages were highly dynamic and changed substantially at both the OTU level and higher taxonomic classifications during time frames consistent with many oceanographic methods used for measuring biological rates. Changes were most dramatic in enrichments, which yielded community compositions that were strongly dominated by one or a few taxa. Changes in community structure during incubation dramatically influenced estimates of species richness, which were substantially lower with longer incubation and especially with amendment, even though all incubated samples originated from the same aliquot of seawater. Containment and enrichment of the seawater sample led to the detection of otherwise undetected protistan taxa, suggesting that characterization of protistan diversity in a sample only at the time of collection could lead to an underrepresentation of unique taxa. Additionally, the rapid increase in the relative abundance of some members of the "rare biosphere" in our results implies an ecological importance of at least some of the taxa comprising the "rare biosphere."

Bacteria in beach sands: an emerging challenge in protecting coastal water quality and bather health.

To protect bather health at recreational beaches, fecal indicator bacterial standards are used to monitor water quality, and waters exceeding the standards are subsequently closed to bathers. However beachgoers are also in contact with beach sands, the sanitary quality of which is not included within beach monitoring programs. In fact, sands and sediments provide habitat where fecal bacterial populations may persist, and in some cases grow, in the coastal zone. Specific pathogens are less well studied in beach sands and sediments, but there is a body of evidence that they too may persist in these environments. This paper reviews the current state of knowledge regarding the abundance and distribution of fecal indicator bacteria and pathogens in beach sands of diverse climatological regions, and at beaches subjected to varied levels of anthropogenic impact. In all regions fecal indicator bacteria are nearly ubiquitous in beach sands, and similar relationships emerge among fecal indicator abundance in dry sand, submerged sands, and water. Taken together, these studies contextualize a potential public health issue and identify research questions that must be addressed in order to support future policy decisions.

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.