Additional file 2 of Microeukaryotic gut parasites in wastewater treatment plants: diversity, activity, and removal
Creators: name: Freudenthal, Jule
name: Ju, Feng
name: Bürgmann, Helmut
name: Dumack, Kenneth
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Pharmacology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: Ecology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Supplementary Figure 1. Comparing microbial communities between WWTP locations to identify outliers. Graphs showing multivariate dispersion (A) and beta diversity (B) of microbial community composition at N=11 WWTP locations, based on metagenomic data (left-hand side) and metatranscriptomic data (right-hand side). For multivariate dispersion (A), NMDS plots were calculated based on Bray-Curtis dissimilarities. Lines (color-coded by location) link samples at each location to their centromere. For beta diversity (B), boxplots show the 25 % and 75 % percentiles and medians of Bray-Curtis dissimilarities. Points are color and symbol-coded by WWTP compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). In beta diversity based on metatranscriptomic data (lower row, right-hand side), significant differences between location “FD” and the other locations are indicated with asterisks (unpaired two-sample Wilcoxon test, * p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001). Based on these results, location FD was excluded from further analysis. Supplementary Figure 2. Assessment of the variation caused by sampling processing (sequencing). NMDS plot based on Bray-Curtis dissimilarities derived from metatranscriptomic data, comparing microbial community composition across WWTP compartments and locations. The variation caused by sample processing is shown for one location, “ZR”, showing three sequencing replicates from the inflow (INF) (replicates indicated by yellow asterisks). Based on this comparison, we concluded that variation caused by sequencing was low. Compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). Supplementary Figure 3. Rarefaction curves for metagenomic (rDNA) and metatranscriptomic (rRNA) data. Curves showing the number of reads as a function of the number of OTUs identified (N=37 samples, i.e. one sample from each WWTP compartment (4) at each WWTP location (10), excluding 3 samples because of exceptionally low sequencing-depth). Samples are color-coded by compartment: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). Supplementary Figure 4. Total number of rDNA and rRNA sequences. Boxplots showing the 25 % and 75 % percentiles and medians of the total number of rDNA (metagenomics) and rRNA (metatranscriptomics) sequences for (A) the total community and (B) the parasitic community, comparing prokaryotes (blue) and eukaryotes (yellow). Compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). Supplementary Figure 5. Microbial community structure and environmental factors across WWTPs. NMDS biplots based on Bray-Curtis dissimilarities showing microbial community composition across WWTP compartments and locations, in association with environmental data. Metagenomic and metatranscriptomic data are shown separately. Samples are color-coded and grouped (ellipses) by compartment. Significant environmental vectors are shown as arrows (* p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001). Compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). Environmental vectors: DO = dissolved oxygen, DOC = dissolved organic carbon, HRT = Hydraulic retention time, TN = total nitrogen, TP = total phosphorus. Supplementary Figure 6. Microbial community structure and environmental factors in the separate WWTP compartments. NMDS biplots based on Bray-Curtis dissimilarities, showing microbial community composition in association with environmental data for each WWTP compartment. Metagenomic and metatranscriptomic data are shown separately. The distribution of the samples (symbol-coded by WWTP location) is visualized by the ellipses. Significant environmental vectors are shown as arrows. Compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent (treated water). Environmental vectors: TN = total nitrogen. Supplementary Figure 7. Co-occurrence networks of parasitic orders in the four WWTP compartments. Networks showing correlations derived from co-occurrence network inferences for each WWTP compartment, based on metagenomic (first row) and metatranscriptomic (second row) data. Only associations that involve parasites are shown. Nodes represent genera grouped at the order level and trait level (red nodes: parasitic taxa; yellow nodes: free-living taxa), with node size proportional to the total number of reads for each order. Edges represent correlations between taxa (blue lines: positive correlations; red lines: negative correlations), with line thickness proportional to the number of genera per order involved. Compartments: INF = inflow (sewage), DNF = denitrification bioreactor, NFC = nitrification bioreactor, EFF = effluent. Abbreviations for Archaea: E = Euryarchaeota, O = Others. Abbreviations for bacteria: A = Actinobacteria, B = Bacteroidetes, C = Chloroflexi, F = Firmicutes, O = Others, Pl = Planctomycetes, Pr = Proteobacteria, T = Tenericutes, V = Verrucomicrobia. Abbreviations for Proteobacteria: Alpha = Alphaproteobacteria, Beta = Betaproteobacteria, Gamma = Gammaproteobacteria, Delta = Deltaproteobacteria, O = Others. Abbreviations Fungi: Ascomycota = A, Basidiomycota = B, Others = O. Abbreviations for protists: Ce = Cercozoa (*including Rosculus), Ci = Ciliophora, Co = Conosa, Db = Discoba, Di = Discosea, Ms = Mesomycetozoa, Mt = Metamonada, O = Others, S = Stramenopiles, T = Tubulinea. Supplementary Table 1. Microbial community composition after quality filtering. Total number and relative number (%) of ribosomal reads and OTUs in the metagenomic (rDNA) and metatranscriptomic (rRNA) data of 10 WWTP locations, for prokaryotes (bacteria and Archaea) and eukaryotes (protists, fungi and microscopic metazoa). Supplementary Table 2. Parasitic genera in WWTPs based on both metagenomic and metatranscriptomic data. Overview of all parasitic genera identified in the WWTP samples. Supplementary Table 3. Comparing the total number of rDNA and rRNA sequences. Pair-wise comparison of the total number of eukaryotic and prokaryotic sequences for the total community and the parasitic community, contrasting the inflow (INF) with the denitrification bioreactor (DNF), the denitrification bioreactor (DNF) with the nitrification bioreactor (NFC), and the nitrification bioreactor (NFC) with the effluent (EFF). Sign test (* p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001). Supplementary Table 4. Comparing the abundance of parasitic protists between WWTP compartments. Pair-wise comparison of relative abundances of parasitic protist taxa in metagenomic and metatranscriptomic data, contrasting the inflow (INF) with the denitrification bioreactor (DNF), and the nitrification bioreactor (NFC) with the effluent (EFF). Sign test (* p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001). Supplementary Table 5. Overview of the most numerous orders in WWTPs. Overview of the most numerous orders shown in Fig. 1. Numbers show their mean relative abundances across all compartments and locations (total N=40 samples) for both rDNA and rRNA data, as well as the absolute difference between these relative DNA and RNA abundances, per order.