Patterns of protist diversity associated with raw sewage in New York City

Abstract

Protists are ubiquitous components of terrestrial and aquatic environments, as well as animal and human microbiomes. Despite this, little is known about protists in urban environments. The ~7400-mile sewer system of New York City (NYC) collects human waste from ~8 million human inhabitants as well as from animals, street runoff, and groundwater, providing an ideal system to study these microbes. We used 18S rRNA amplicon sequencing and shotgun metagenomic sequencing to profile raw sewage microbial communities. Raw sewage samples were collected over a 12-month period from 14 treatment plants of the five NYC boroughs, and compared with samples from other environments including soil, stormwater, and sediment. Sewage contained a diverse protist community dominated by free-living clades, and communities were highly differentiated across environments. Seasonal differences in protist composition were observed; however, network analysis and functional profiling demonstrated that sewage communities were robust and functionally consistent. Protists typically associated with human and animal guts or feces were frequently detected. Abundance of these parasites varied significantly both spatially and temporally, suggesting that spikes could reflect trends in the source population. This underscores sewage as a valuable model system for monitoring patterns in urban microbes and provides a baseline protist metagenome of NYC.

Fig. 1

Geographical location of samples. Colors represent DEP drainage areas, and each number represents one of the 17 raw sewage samples. Black lines show NYC borough boundaries; white shapes indicate locations of samples from other environments; triangle = private building; square = soil; diamond = sediment. DEP drainage areas were adapted from the official NYC DEP plant map: http://www.nyc.gov/html/dep/html/wastewater/wwsystem-plantlocations_wide.shtml

Fig. 2

Beta diversity and LEfSe enrichment of protist taxa in five different environments. a, b V4 region. c, d V9 region. Beta diversity ordinations (a, c) were calculated from NMDS of the Bray–Curtis dissimilarity. Cladograms (b, d) show significant associations of protist taxa with an environment; each of the five environments is represented by a different ring. In all panels colors represent the environment from which each sample was collected, and shape represents the collection site; shading is proportional to the LDA score

Fig. 3

Relative abundance of protist groups identified in DEP and private sewage collected in November 2014. a V4 region. b V9 region. In both panels data are the average of two replicate samples. Colored horizontal bars indicate DEP (light blue) and private sewage (dark blue) samples. Dotted lines separate samples by borough. Abbreviations: Bk = Brooklyn, BQ = Brooklyn/Queens, Qu = Queens, SI = Staten Island, Bx = Bronx, BM = Bronx/Manhattan, M = Manhattan

Fig. 4

MetaCyc pathways inferred from shotgun metagenomic sequence data in November 2014 DEP sewage samples. Rows represent MetaCyc pathways identified with HUMAnN2, columns represent samples, and cells are colored according to the number of sum-normalized reads per kilobase on a log scale. Members of the most abundant pathway classes are indicated by colored bars to the right of the heat map. Shapes represent the borough from which each DEP sample was collected. Abbreviations: Bk = Brooklyn, BQM = Brooklyn/Queens/Manhattan, BQ = Brooklyn/Queens, Qu = Queens, SI = Staten Island, Bx = Bronx, BM = Bronx/Manhattan, M = Manhattan

Fig. 5

Diversity of DEP sewage over time. Both beta diversity ordinations are from NMDS of the Bray–Curtis dissimilarity for all 136 DEP samples by season and borough. a V4 region. b V9 region. In both panels colors indicate collection season and shapes represent the borough from which each sample was collected. Abbreviations: Bk = Brooklyn, BQ = Brooklyn/Queens, Qu = Queens, SI = Staten Island, Bx = Bronx, BM = Bronx/Manhattan, M = Manhattan. Relative abundance of protist groups identified in DEP sewage over four seasons from 18S rRNA amplicon sequencing. c V4 region. d V9 region. In both panels data are the average of all 34 samples collected per season

Fig. 6

Relative abundances of genera of interest in DEP sewage samples over time from 18S rRNA gene amplicon sequencing. For all heat maps rows indicate collection season, columns represent samples, and each cell shows the average relative abundance from two replicate samples. a Entamoeba relative abundance. b Blastocystis relative abundance. c Trichomonas relative abundance. d Tritrichomonas relative abundance. In all panels lines separate samples by borough. Abbreviations: Bk = Brooklyn, BQ = Brooklyn/Queens, Qu = Queens, SI = Staten Island, Bx = Bronx, BM = Bronx/Manhattan, M = Manhattan