Research on Harmful Algal Blooms Jorge W. Santo Domingo, ORD/CESER.WID/BCB [email protected]
Ongoing Projects:
• Identification of cyanobacterial species, their population dynamics and levels of activity in Lake Harsha using 16S rRNA gene sequencing approaches. Generated and analyzed molecular data for three years (2015-2019). Major cyanobacterial bacterial species identified and their level of activity determined.
• Development of 18S rRNA gene database to identify eukaryotic planktonic community Generated and analyzed molecular data for three years (2015-2017). Eukaryotic groups have been described and compared with
• Use of regression models to determine the planktonic biota that is associated to microcystin blooms Several bacterial and eukaryotic taxonomic groups and environmental and biological factors showed + or - relationships
• Identification of bacterial taxa that can be used in forecasting cyano bacterial blooms
• Evaluation of commercially available kits for the detection of microcystin, anatoxin, and saxitoxin in Lake Harsha and in different Ohio drinking water sources
• Molecular survey of cyanobacterial species in Ohio drinking water sources • DISCLAIMER: The views expressed in this presentation are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. Taxonomy based on 16S rRNA gene sequences
Cyanobacterial genera in Lake Harsha
p__Cyanobacteria
c__Nostocophycideae g__Aphanizomenon g__Cylindrospermopsis g__Dolichospermum g__Trichormus g__Anabaena c__Oscillatoriophycideae g__Microcystis g__Phormidium g__Planktothrix g__Snowella c__Synechococcophycideae g__Pseudanabaena g__Synechococcus HAB Monitoring Tools
HF Physico-chemical Phototroph In-vivo Cyanotoxin Molecular Methods Wet Chemistry Fluorescence • Temperature • Toxicity Using DNA and RNA as templates to • Total Nitrogen • pH • BBE Algae Online – Online Toxicity determine the presence and level of Analyzer activity of different groups • NO -NO • ORP Monitors 2 3 – Green Algae • Next Gen Sequencing • Total NH • Specific Conductance – Laboratory Assays 4 – 16S rRNA gene • Turbidity – Cyanobacteria • Analytical • Total Phosphorous Quantification – 18S rRNA gene • Dissolved Oxygen – Brown (diatoms) • Total Reactive – Cryptophyta – ELISA – Cytochrome oxidase I • Total Organic Carbon – Metagenomie Phosphorous – Total Chlorophyll • MC-ADDA • Dissolved Organic – Metatranscriptome Carbon • YSI – LC-MSMS • Microcystin • PCR and qPCR assays • NO3-N – Total Chlorophyll congeners – Toxin specific gene • UV-Vis spectral profile – Phycocyanin assays • PAR • Cylindrosper (cyanobacteria) mopsin – 16S rRNA gene group • Weather • Anatoxin-a and genus specific assays • MMPB – 18S rRNA gene group specific assays The phylogenetic relationships of cyanobacteria inferred from 16S rRNA (A), rbcL (B), and hetR (C) nucleotide sequences; trees were constructed by the ML method (19).
Akiko Tomitani et al. PNAS 2006;103:14:5442-5447
©2006 by National Academy of Sciences Drawbacks of DNA-based methods: • Under some environmental conditions DNA can resist degradation • Associated with cell debris and dead cells • Their correlation with viable/active bacteria can be shaky at times • Does not strictly imply presence of bacteria recently inhabiting a given matrix Omics Central Metabolomics
Metabolites
DNA RNA Protein
Genomics Transcriptomics Proteomics Metatranscriptomics Metagenomics RNA-based methods:
• RNA more unstable than DNA in the environment due to RNAses • Declines quickly in less active cells, thus correlates with cellular activity • Natural amplification of targeted gene, i.e., 1000s to 10000s of copies per cell • Better correlation with active (viable?) bacterial fraction Most detected targets belong to active bacteria?
Figure 3 Lake Harsha, Clermont County, OH
• Watershed Area • 342 miles2 • Summer Pool Elevation • 733 ft • Summer Pool Area • 2000 acres • Water Quality Data since 2012 at 3 week intervals Environ. Sci. Technol. 2013, 47:13611–13620 0.2k__Bacteria 0.2.1 p__AC1 k__Bacteria taxon 0.2.4 p__Actinobacteria p__Cyanobacteria 0.2.7 p__Armatimonadetes k__Bacteria c__Chloroplast 0.2.9 p__BRC1 Taxa identified using 16S rRNA gene o__Chlorophyta 0.2.10p__Bacteroidetes p__Cyanobacteria sequencing data f__Chlamydomonadaceae 0.2.15p__Chlorobi c__4C0d-2 0.2.16p__Chloroflexi o__Stramenopiles 0.2.18p__Cyanobacteria c__Chloroplast o__Cryptophyta 0.2.23p__Elusimicrobia o__Euglenozoa c__ML635J-21 0.2.24p__FBP o__Haptophyceae 0.2.26p__Fibrobacteres c__Nostocophycideae o__Streptophyta 0.2.27p__Firmicutes c__Nostocophycideae 0.2.28p__Fusobacteria c__Oscillatoriophycideae g__Aphanizomenon 0.2.31p__GN02 c__Synechococcophycideae 0.2.34p__Gemmatimonadetes g__Cylindrospermopsis 0.2.35p__H-178 unclassified g__Dolichospermum 0.2.41p__Lentisphaerae g__Trichormus 0.2.45p__NC10 g__Anabaena 0.2.46p__NKB19 c__Oscillatoriophycideae 0.2.48p__Nitrospirae g__Microcystis 0.2.50p__OD1 g__Phormidium 0.2.52p__OP11 g__Planktothrix 0.2.53p__OP3 g__Snowella 0.2.54p__OP8 0.2.58p__Planctomycetes c__Synechococcophycideae 0.2.60p__Proteobacteria g__Pseudanabaena 0.2.64p__SR1 g__Synechococcus 0.2.65p__Spirochaetes 0.2.68p__TM6 0.2.69p__TM7 0.2.71p__Tenericutes 0.2.72p__Thermi 0.2.75p__Verrucomicrobia 0.2.77p__WS1 0.2.79p__WS3 0.2.80p__WS4 0.2.81p__WS5 0.2.84p__ZB3 Dominant bacterial taxa in Lake Harsha 2015
BUOY 60.00 Actinobacteria Actinobacteria JSD Cyanobacteria Cyanobacteria JSD
50.00
40.00 escneuseq
30.00 A DNal ot DNal A
20.00 t % of t
10.00
0.00
Sampling Dates Dominant Cyanobacterial Classes vs Algal Aduncance
60.00 p__Cyanobacteria c__Chloroplast c__Nostocophycideae c__Oscillatoriophycideae c__Synechococcophycideae
50.00
40.00
30.00 % of total seqeunces oftotal % 20.00
10.00
0.00
Sampling Dates Cyanobacterial trends using DNA vs RNA (cDNA) as targets for sequencing libraries EMB p__Cyanobacteria-cDNA p__Cyanobacteria-DNA 100
90
80
70
60
50
40 % of total sequences % oftotal 30
20
10
0
Sampling Dates EFLS 100.00 p__Cyanobacteria-DNA p__Cyanobacteria-cDNA
90.00
80.00
70.00 escenuseqal ott % of ott escenuseqal 60.00
50.00
40.00
30.00
20.00
10.00
0.00
Sampling Date EFLD 90.00 p__Cyanobacteria-DNA p__Cyanobacteria-cDNA
80.00
70.00
escenuseqal ott% of ott% escenuseqal 60.00
50.00
40.00
30.00
20.00
10.00
0.00
Sampling Sites BUOY DNA vs microcystin 2015 60.00 3.500 g__Cylindrospermopsis g__Dolichospermum g__Microcystis g__Planktothrix MC-total
3.000 50.00 saders ecneuqal seot t% of t% seot ecneuqal saders Microcystin 2.500 40.00
2.000
30.00 mg/L 1.500
20.00 1.000
10.00 0.500
0.00 0.000
Sampling Dates BUOY RNA vs microcystin 2015 100.00 3.500 g__Aphanizomenon g__Cylindrospermopsis g__Dolichospermum g__Microcystis g__Planktothrix MC-total
90.00 3.000 80.00 Microcystin
70.00 2.500
60.00 2.000
50.00 mg/L 1.500 40.00
30.00 1.000
% or total sequences reads sequences total % or 20.00
0.500 10.00
0.00 0.000
Sampling Dates Microcystis Cyclindrospermopsis Lake Harsha 2015. Sphingobacteriales Bacillariophyta Random forest regressions Saprospirales Candidatus Xiphinematobacter featuring: Alphaproteobacteria ACK-M1 (Actinomycetales) • 88 eukaryotic (18S) OTUs • 142 prokaryotic (16S) OTUs Flavobacterium Chitinophagaceae • 12 different abiotic Synechococcus environmental Actinomycetales measurements (including Cercozoa lake inflow/outflow, temperature, pH, rainfall, Saprospiraceae (Saprospirales) Pseudanabaena and N and P concentrations). Burkholderiales
Agaricomycotina
Pseudanabaenaceae 211ds20 (Alteromonadales) Bacteroidetes Higher variance explained for MC-LR; same taxa , slightly different rankings Random Forest Regression Model: Predict Microcystis Relative Abundance
4-day Interval Microcystis Variance Most informative Taxonomy Explained Model Feature Same interval 90.1% OTU275 Deltaproteobacteria, Myxococcales 1 prior 78.0% OTU317 Betaproteobacteria 2 prior 84.8% OTU196 Bacteroidetes 3 prior 90.1% OTU45 Acetobacteraceae, Roseomonas 4 prior 77.3% 3-day ave. rain Forest regression for Lake Harsha 2015 using 16S rRNA gene sequencing data Ohio EPA and USEPA Interlab Phytoxigene Assay (16S) Method Comparison 1000000
100000 R² = 0.6943
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1
0.1 0 1 10 100 1000 10000 100000 1000000 Any questions?