Dataset Information

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

ABSTRACT: Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

SUBMITTER: Maritz JM

PROVIDER: S-EPMC5653731 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Maritz Julia M JM Rogers Krysta H KH Rock Tara M TM Liu Nicole N Joseph Susan S Land Kirkwood M KM Carlton Jane M JM

Microbial ecology 20170524 4

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of pro ...[more]

PMID: 28540488

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Project description:For photosynthetic microbial eukaryotes, the rate-limiting step in NO3- assimilation is its reduction to nitrite (NO2-), which is catalyzed by assimilatory nitrate reductase (NR). Oceanic productivity is primarily limited by available nitrogen and, although nitrate is the most abundant form of available nitrogen in oceanic waters, little is known about the identity of microbial eukaryotes that take up nitrate. This lack of knowledge is especially severe for ice-covered seas that are being profoundly affected by climate change. To address this, we examined the distribution and diversity of NR genes in the Arctic region by way of clone libraries and data mining of available metagenomes (total of 4.24 billion reads). We directly compared NR clone phylogenies with the V4 region of the 18S rRNA gene (DNA pool) and 18S rRNA (RNA pool) at two ice-influenced stations in the Canada Basin (Beaufort Sea). The communities from the two nucleic acid templates were similar at the level of major groups, and species identified by way of NR gene phylogeny and microscopy were a subset of the 18S results. Most NR genes from arctic clone libraries matched diatoms and chromist nanoflagellates, including novel clades, while the NR genes in arctic eukaryote metagenomes were dominated by chlorophyte NR, in keeping with the ubiquitous occurrence of Mamiellophyceae in the Arctic Ocean. Overall, these data suggest that a dynamic and mixed eukaryotic community utilizes nitrate across the Arctic region, and they show the potential utility of NR as a tool to identify ongoing changes in arctic photosynthetic communities.IMPORTANCE To better understand the diversity of primary producers in the Arctic Ocean, we targeted a nitrogen cycle gene, NR, which is required for phytoplankton to assimilate nitrate into organic forms of nitrogen macromolecules. We compared this to the more detailed taxonomy from ice-influenced stations using a general taxonomic gene (18S rRNA). NR genes were ubiquitous and could be classified as belonging to diatoms, dinoflagellates, other flagellates, chlorophytes, and unknown microbial eukaryotes, suggesting novel diversity of both species and metabolism in arctic phytoplankton.

Dataset Information

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Publications

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Similar Datasets

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