Both coastal inlets and treated wastewater outfalls are recognized as major pathways for microbial contaminants from Land-Based Sources of Pollution (LBSP) to enter Florida's Southeastern coastal marine ecosystems, including an area containing reef tracks with an estimated annual economic value in excess of $3.88 Billion. These LBSP discharges can contain a variety of microbial contaminants including fecal indicator bacteria, fecal host marker bacteria, and pathogens to both human health and coral ecosystem health. The study reported here presents the first year of data from a two-year CRCP project (Project #1114), as part of the broader NOAA Florida Area Coastal Environment (FACE) research program and is established as the first preliminary pilot study for the broader NOAA Coral Genomic Observatory Network (CGON). This project is studying prevalence of fecal indicator bacteria, molecular microbial source tracking markers for fecal contaminants, selected pathogenic bacteria, as well as characterizing the bacterial, algal, and fungal communities of coastal inlet discharges, oceanic wastewater discharges, coral reef waters, and of actual coral tissue from 4 sentinel reefs in this study area offshore of Miami-Dade and Broward Counties in Florida. The data files referred to here that are archived at CORIS and at AOML, and linked to this year 1 metadata file include: (1) an excel spreadsheet file of the sample inventory log listing samples from year 1 with the dates, locations, sample types, unique filter ID # for extractions, processing status, and which samples have been sequenced; (2) an excel spreadsheet file for the viable enterococci data from the year 1 samples giving live enterococci counts in cfu/100mL water per EPA method 1600 for water samples from coastal inlets, wastewater outfalls, wastewater effluent, and coastal waters from sentinel reef sites; (3) an excel spreadsheet file showing data for year 1 water samples and selected year 1 coral tissue samples showing molecular microbial source tracking qPCR results for total enterococci, for Bacteroidales fecal markers specifically associated with cow, dog, and human hosts, and qPCR data for Human Polyoma Virus and for Pepper Mottled Mild Virus (both viral human fecal markers), and a summary of microfluidic qPCR analysis for specific food-borne and water-borne pathogen genes in selected water and coral tissue samples; (4) an excel spreadsheet showing nutrient and physical data and metadata for water samples, including GPS locations of water samples, dates, times, CTD cast numbers, depth, temperature, salinity, conductivity, density, CDOM, turbidity, oxygen saturation, and nutrients (N+N, TKN, TP, Chlorophyll), also includes embedded google earth map images showing sample sites; (5) an excel spreadsheet showing the physical data, observations, and metadata from divers diving computer for the coral benthic observations, including site GPS location, time, date, depth, temperature, and coral biometric observations, also includes embedded map images of coral/algal observation sites, and embedded charts of coral bleaching; (6) a powerpoint file showing a summary of the sequencing and metagenomic bioinformatics data, including summary of sequencing and bioinformatics analysis methods, rarefaction plots, heat maps of most prevalent phyla, heat maps of most prevalent families, PCoA plots comparing populations of coastal inlets, wastewater outfalls, and coral reefs, PCoA plots for populations of coral tissues, and preliminary conclusions so far for year 1 data. The actual Illumina Sequence data for each sample is archived at the National Center for Biotechnology Information (NCBI) as Bioproject # 292127 (see link above). This bioproject sequence data is scheduled to be available to the public on May 31, 2016. Until this time, access to the sequence files is password protected. If you require access to these files before this date, please contact the project PI, Dr. Christopher Sinigalliano (email: christopher.sinigalliano@noaa.gov).
The main objectives of this project on coral reef LBSP microbial source tracking and reef community microbial metagenomics is to measure specific microbial contaminants from land based sources of pollution within the water column of coastal inlets, outfalls, and coral reef tracks of the Southeast Florida region, and within the coral itself of four regional reefs. Specific pathogens and microbial source tracking markers are measured by quantitative PCR, coral symbiont algae populations are genetically characterized, and microbial diversity and community composition is determined by metagenomic sequencing analysis of bacteria, fungal, and algal populations. This project is expected to aid management with new molecular tools to help identify and mitigate sources of land based microbial contaminants impacting coral reefs and to provide information on the ecosystem microbial community structure to better assess reef ecosystem health. We report here the Year 1 efforts of this 2 year project (leveraged with on-going AOML field collection and benthic coral survey programs) to collected bimonthly water samples and quarterly coral samples for molecular microbial analysis of LBSP bacterial contaminants by both target specific qPCR methods and by total microbial community next-generation sequencing and metagenomic analysis, and to characterize coral symbiont populations by qPCR. Correlating patterns of pollutant exposure with microbial community composition may help identify threshold levels of exposure, and PCR source tracking may identify originating host sources to discriminate between sewage/septic, agricultural, urban runoff, etc., thus aiding in targeted mitigation efforts.
Types of data collected and Metagenomic Community Sequencing: This molecular characterization consists of next-generation total community sequencing with ribosomal primers for bacterial, fungal, and algal populations, metagenomic bioinformatics analysis of this NGS data, along with viable plate count data for live enterococci fecal indicaotrs (by EPA method 1600), qPCR based source tracking of human-host Bacteroidales, canine-host Bacteroidales, bovine-host Bacteroidales, total enterococci by EPA method 1611, and Symbiodinium clade characterization of coral symbiont populations for the coral tissue samples by qPCR. Additional data presented here that was collected as part of the larger FACE program includes water quality nutrient data and physical measurements for depth, temperature, salinity, conductivity, density, CDOM, turbidity, oxygen saturation, and nutrients (N+N, TKN, TP, Chlorophyll). Coral tissue samples were collected by divers as part of a broader Coral Benthic Survey Program by AOML, and that benthic survey data is also presented here including depth, temperature, coral/algal ratios, and biometric data on coral coverage and bleaching. Water samples were collected by CTD cast in Niskin bottles during FACE water quality cruises. Coral tissue samples were collected by divers using sryinge biopsy during quarterly coral benthic surveys.
in progress
NOAA requests that all individuals who use NOAA data acknowledge the source of these data in any reports, papers, or presentations. If you publish these data, please include a statement similar to: Some or all of the data described in this article were produced by the NOAA's Atlantic Oceanographic and Meteorological Laboratory via its Coral Genomic Observatory Network and Florida Area Coastal Environment Program
4301 Rickenbacker Causeway
This data set was developed by National Oceanographic and Atmospheric Administration (NOAA), Atlantic Oceanogrpahic and Meteorological Laboratory, Coral Genomic Observatory Network as part of Coral Reef Conservation Project # 1114
QA/QC routines for nutrient data, MST data, and sequencing data that flags data outside of the range of what are considered to be valid values for that parameter.
At the current time, this represents year 1 data only, the analysis and data are not complete yet. All project sample collection for year 1 and year 2 has been completed as of October 1, 2015, but additional metagenomics analysis is still on-going for both year 1 and year 2 samples. Data completion is scheduled for May 31, 2016.
Data Acquisition and Sample Collection: The sample collection methods used by NOAA and NOAA contractor field crews will be described here. (1): Water sample collection - Surface water samples exposed to point and non-point sources of wastewater pollution were collected during bimonthly Numeric Nutrient Criteria research cruises aboard the U.S. NOAA Ship Hildebrand from November 2013 through July 2015 for the data presented here as part of the Florida Area Coastal Environment (FACE) Program. Physical water samples were collected by CTD casts using rosette of sanitized 10-liter Niskin bottles, with CTD sensors for salinity, temperature, depth, conductivity, pH, fluorescence, and oxygen saturation. Physical environmental data, nutrient data, and metadata were generated by the FACE program. Surface water samples exposed to WWTP effluent were collected at the Miami-Dade North WWTP and Miami-Dade Central WWTP ocean outfall surface boils (N25.923061, W-80.089369; N25.742817, W-80.085967, respective), Surface water samples, potentially exposed to malfunctioning septic systems, were also collected during the outgoing tide from the following inlets: Port Everglades (N26.093450, W80.109722), Port of Miami (N25.763611, W80.132778), and Bakers Haulover (N25.900000, W80.121389). Coastal water was also collected offshore to serve as background process controls during each sampling event. Water samples were also collected from three sites each from four reefs: Emerald Reef, Barracuda Reef, Pillars Reef, and Oakland Ridge Reef. All water samples were collected from CTD Niskin bottles into 2-liter sterile polypropylene bottles and were transported on ice during sampling and returned to lab and maintained at 4C until filtration. All water samples were filtered within 24 hours of collection. (2) Coral Tissue Samples: Coral polyp tissue and mucus with its associated microbiome was collected during quarterly coral benthic survey dives by NOAA contractor research divers from three sites each of four reefs: Emerald Reef, Barracuda Reef, Pillars Reef, and Oakland Ridge Reef. Two separate coral species, Siderastrea siderea and Porites astreoides, were collected for each coral sample site. Coral tissue was collected by syringe polyp biopsy, pressing a 5-mL syringe closely against the coralite skeleton and withdrawing the plunger sharply, sucking the polyp into the syringe. Replicate polyps near each other from the same head were collected in the syringe, and upon return to the boat were pooled together in 95% ethanol in a 50mL Falcon tube. Coral tissue samples were kept in 95% ethanol preservative on ice until return to the lab, then stored at -20 degrees C until DNA extraction.
Data Acquisition and Sample Processing: The sample processing and analysis methods will be described here. (1): Water sample processing for molecular analysis - one liter water samples were aseptically filtered onto mixed cellulose ester filters (47mm diameter, 0.2 micron pore size)to collect total particulates greater then 0.2 microns. These filters were aseptically transferred to Lysing Matrix "A" tubes (MP Biomedicals), and total genomic DNA extracted with the FastDNA Spin Kit (MP Biomedicals) as permanufacturers instructions using the CLS-VF lysis buffer and a FastPrep-24 bead beat homogenizer (MP Biomedicals), using one round of bead beating for 60 seconds at a speed setting of 6.0 mps. Aliquots of purified eluted DNA was stored at -20 degrees C for later analysis. Aliquots of purified eluted DNA for long-term cryoarchiving was stored at -80 degrees C. (2): Coral tissue sample processing for molecular analysis - Coral polyp tissue preserved in 95% ethanol was harvested from the ethanol by filtration on polycarbonate membrane filters (47mm diameter, 0.2 micron pore sized), then the filters with coral tissue aseptically transferred to Lysing Matrix "E" tubes (MP Biomedicals). Total genomic DNA from the filters (including both coral and the associated microbiome) was extracted using the FastPrep DNA Spin Kit for Soil kit (MP Biomedicals) as per manufacturer's directions with the following modifications. Two rounds of bead beating were conducted with a FastPrep-24 bead beat homogenizer (MP Biomedicals) for 60 seconds each at a speed setting of 6.0 mps. The eluted genomic DNA from coral samples was stored as described above for water sample DNA.
Sample Analysis and Data Preparation: A summary of sample analysis methods and Data Preparation are described here. (1): analysis and enumeration of live enterococci fecal indicating bacteria, normalized to units of colony forming units per 100mL, were conducted by mEI agar viable plate counts as per EPA method 1600 for viable enterococci. (2): Quantitative PCR analysis for Microbial Source Tracking - the qPCR for source tracking of total enterococci, human, and dog specific Bacteroidales was as per Sinigalliano et al (2010, Water Research, 44:3763-3772), qPCR for cow specific Bacteroidales was as per Shanks et al (2008, Applied Environmental Microbiology, 74:745-752), qPCR for PMMoV and HPyV viruses was as per Symonds et al (2014, Water Research, 65:267-270). QC controls included field blanks, sample processing blanks, qPCR extraction controls, qPCR inhibition controls, enterococci bioball positive controls, etc.) (3): Next-Generation-Sequencing of metagenomic community DNA by Illumina chemistry - DNA extracts were normalized to a concentration of 2.5 ng ae-1 by dilution in nuclease-free water and used as template for a PCR assay targeting the hypervariable V4 region of the 16S rDNA. Each sample was amplified with a reverse primer containing a unique 6 bp nucleotide sequence on the 5'-terminus to allow samples to be pooled for sequencing and separated later. Library construction and paired-end sequencing (2 x 100 nt) of amplicons was performed by the BioMedical Genomics Center at the University of Minnesota (Saint Paul, MN) using the Illumina HiSeq platform (U. Minn. PI: Chanlan Chun). (4): Bioinformatics analysis and data preparation - Raw sequence reads were pair-end aligned, separated by sample, and trimmed for quality using Mothur software v.1.36.0. Sequences of abundance <2 over the entire dataset were excluded from analysis. Sequences were aligned to the RDP taxonomic database, and analysis of OTUs was performed at a sequence cutoff of 0.02. The number of sequence reads associated with each group was subsampled to that of the smallest group for comparisons of beta diversity (total species diversity and turnover among samples) and relative taxonomic abundance. Alpha diversity indices (Chao, Ace, Shannon, non-parametric Shannon, and Simpson indices) were calculated using Mothur. The Bray-Curtis measure of dissimilarity was used to construct distance matrices among sites. Weighted and unweighted UniFrac analyses were also calculated using Mothur software package.
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