http://lod.bco-dmo.org/id/dataset/661973
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2016-10-19
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Accession numbers and images of coral samples taken in the Florida Keys during 2013 (Coral Microbial Relationships project)
2016-10-19
publication
2016-10-19
revision
BCO-DMO Linked Data URI
2016-10-19
creation
http://lod.bco-dmo.org/id/dataset/661973
Amy Apprill
Woods Hole Oceanographic Institution
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Apprill, A. (2016) Accession numbers and images of coral samples taken in the Florida Keys during 2013 (Coral Microbial Relationships project). Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version Final) Version Date 2016-10-19 [if applicable, indicate subset used]. http://lod.bco-dmo.org/id/dataset/661973 [access date]
Species images and accession numbers for tissue, mucus, and holobionts analyzed. Dataset Description: <p>&nbsp;</p>
<p>Partial SSU rRNA genes of bacteria and archaea from coral mucus, tissue and holobiont samples produced using 515F/806R primers.</p> Methods and Sampling: <p><strong>Sample collections:&nbsp;</strong></p>
<p>Triplicate colonies of the corals&nbsp;Orbicella&nbsp;(formerly&nbsp;Montastrea)&nbsp;faveolata&nbsp;(Budd et al., 2012),&nbsp;Montastrea&nbsp;cavernosa,&nbsp;Diploria&nbsp;strigosa,&nbsp;Porites&nbsp;astreoides&nbsp;and&nbsp;Porites porites&nbsp;were sampled via SCUBA diving using hammer and chisel at four sites within the Florida Keys, offshore of Summerland Key at depths ranging from 2.4 – 7.6 m (Reef flat, 24 deg 33.155’, 81 deg 22.88’; Open water patch reef, 24 deg 33.164, 81 deg 26.225; Mid-channel patch reef 24 deg 33.620, 81 deg 30.076; Nearshore reef, 24 deg 36.341, 81 deg 25.756 and an offshore Nursery site, 24 deg 33.745, 81 deg 24.013, where corals had been transplanted from nearby reefs). Fragments from each colony were placed in sterile Whirl-Pak bags (Nasco, Fort Atkinson WI, USA) underwater and transferred to a cooler containing ice after dive completion.&nbsp; Within 1-4 hours, each fragment was rinsed with 0.1 um filtered seawater. Coral mucus was collected by siphoning the mucus from the coral surface with a pipette, and mucus fractions were frozen at -80 deg C.&nbsp; The coral fragment was then divided using a sterilized chisel and hammer into two smaller fragments; one frozen at -80 deg C and the second placed in a 4% paraformaldehyde-0.2 um filter sterilized phosphate buffered saline solution, fixed at 4 deg C for 1 hour, and stored at -20 deg C.</p>
<p>At each site, seawater temperature, dissolved oxygen, and pH were measured&nbsp;from&nbsp;a depth just above the corals (~5 m) and from the surface (0-1 m) using an EXO water quality sonde (YSI Inc., Yellow Springs, OH, USA).&nbsp; Seawater samples (4 l) from the same depths were also collected for microbial biomass, inorganic nutrient&nbsp;analyses&nbsp;and direct cell enumeration, and stored on ice for 1-4 hours.&nbsp; Upon arrival at the laboratory, water for nutrient analyses was frozen at -20 deg C.&nbsp; For direct cell counts, duplicate 1 ml aliquots of seawater were preserved at a final concentration of 4% paraformaldehyde for 20 minutes at 4 deg C and then frozen at -80 deg C. &nbsp; Finally, ~2 l of water was pressure filtered onto duplicate 25 mm, 0.22 um Durapore membrane filters (Millipore, Boston, MA, USA) using a peristaltic pump for&nbsp;collection&nbsp;of seawater microbial biomass and stored at -80 deg C.</p>
<p><strong>Nutrient and pigment analyses and direct cell&nbsp;counts:&nbsp;</strong></p>
<p>Concentrations of dissolved inorganic nutrients (NH4+, NO3–&nbsp;+ NO2–, NO2-, PO43–, and silicate) were measured using a continuous segmented flow system with methods previously described (Apprill and Rappé, 2011), with NO3-&nbsp;was derived from subtracting the contribution of NO2-.&nbsp;&nbsp;NH4+&nbsp;was also measured in the field on unfrozen samples using the ortho-phthaldialdehyde&nbsp;fluorescence method (Holmes et al., 1999;Taylor et al., 2007).&nbsp; Pigment analysis of the phytoplankton community was performed using&nbsp;high-performance&nbsp;liquid chromatography with known standards on extracts from the frozen filters, and quantified as described previously (Van Heukelem and Thomas, 2001). Seawater microbial cell counts were measured using flow cytometry (Apprill and Rappé, 2011). &nbsp;</p>
<p><strong>Preparation of nucleic acids:</strong>&nbsp;</p>
<p>In order to examine mucus- and tissue-associated microbes, coral samples were processed using three approaches (Fig. 1A). &nbsp; The first approach harvested the mucus that was collected as previously described (hereafter referred to as ‘mucus’ samples). For the second approach, the frozen coral tissue (which still contained some mucus) was removed from the skeleton using an airbrush with 80 psi air pressure and 0.22 um filtered phosphate buffered saline solution.&nbsp; The tissue homogenate was then vortexed for 2 min and centrifuged at 20,000 x g for 20 min at 4 deg C. After removal of the supernatant, the cells were stored at -80 deg C for 1 week or less (referred to as ‘holobiont’ samples).&nbsp; Lastly, the third biomass substrate analyzed was decalcified coral tissue, referred to as ‘tissue’ samples, which were devoid of mucus and skeleton. To obtain these samples, the coral subsample that was initially preserved in paraformaldehyde solution was placed in a 20% EDTA solution (Acros Organics Thermo Fisher Scientific, NJ, USA) for 2-3 weeks at 4C on a slow rocker. The EDTA solution was exchanged daily until complete skeleton dissolution, (similar to Ainsworth et al., 2015).&nbsp;</p>
<p>Nucleic acids were extracted from the seawater membrane filters (1/4 of the 25 mm filter), the&nbsp;holobiont&nbsp;cells, and the decalcified tissue samples using the PowerPlant Pro DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA), with modifications that included adaptations that included 350 mg of 0.1mm silica beads (MP Biomedicals, Irvine, CA, USA) to the bead solution and 25 ul of proteinase K (Qiagen Inc., Valencia, CA, USA) followed by incubation at 65 deg C for 60 min. Because tissue samples were initially preserved in paraformaldehyde, they were subject to an extended&nbsp;30-minute&nbsp;proteinase K digestion at 55 deg C followed by an additional high-heat incubation step at 90 deg C for 60 minutes before&nbsp;beadbeating. The PowerPlant Pro extraction method did not result in&nbsp;high-quality&nbsp;DNA from the mucus.&nbsp; Therefore, the PowerBiofilm DNA Isolation kit (Mo Bio Laboratories), containing inhibitor removal steps but otherwise a similar bead mixture and proteinase K digestion at 65 deg C for 60 min, was used for mucus samples. All nucleic acids were quantified using the Qubit dsDNA BR fluorescent assay (Invitrogen Corp., Carlsbad, CA, USA) on a Qubit 2.0 fluorometer.</p>
<p><strong>Sequencing of&nbsp;V4&nbsp;region of archaeal and bacterial SSU rRNA genes:&nbsp;</strong></p>
<p>Barcoded primers targeting the V4 hypervariable region of the SSU rRNA gene,&nbsp;515F&nbsp;and 806R, were utilized for sequencing analysis as detailed in&nbsp;Kozich&nbsp;and colleagues (2013).&nbsp; This sequencing and data analysis occurred prior to the modifications of these primers to capture additional SAR11 clade bacteria (Apprill et al., 2015) and Thaumarchaeota (Parada et al., 2015), and therefore SAR11 are likely underrepresented by 15-25% in the seawater samples; Thaumarchaeota are probably not heavily underestimated in this study because the archaeal&nbsp;amoA&nbsp;gene abundances suggest their low abundance in reef seawater.&nbsp; Triplicate 25 ul PCR reactions contained 1.25 U of GoTaq Flexi DNA Polymerase (Promega Cooperation, Madison, WI, U.S.A.), 5X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, 200 uM of each dNTP, 200 nM of each barcoded primer, and 0.15 ng - 2.0 ng of genomic template for most samples, with some samples diluted or concentrated to encompass a range of 0.0057 to 4.40 ng. The reaction conditions included an initial denaturation step at 95 ºC for 2 min, followed by 32-39 cycles of 95 ºC for 20 s, 55 ºC for 15 s, and 72 ºC for 5 min, concluding with an extension at 72 ºC for 10 min. The reactions were carried out on a thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction products (5 ul) were screened on a 1% agarose-TBE gel.&nbsp; Samples were optimized for PCR at the lowest number of cycles that resulted in an amplified PCR product detected on a gel with the HyperLadder II standard (generally 5 ng ul-1) (Bioline, Taunton, MA, USA), thus minimizing biases from over-amplification.&nbsp; The three replicate reactions were excised from the gel, combined, and purified using the Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, California, USA), and quantified using the Qubit dsDNA HS fluorescent assay.&nbsp; Barcoded amplicons were pooled in equimolar ratios (5 ng each) and shipped to the University of Illinois W.M. Keck Center for Comparative and Functional Genomics for&nbsp;construction&nbsp;of libraries and sequenced using 2x250 bp paired-end MiSeq (Illumina, San Diego, CA, USA).</p>
<p><strong>References:</strong></p>
<p>Ainsworth, T.D., Krause, L., Bridge, T., Torda, G., Raina, J.-B., Zakrzewski, M., Gates, R.D., Padilla-Gamino, J.L., Spalding, H.L., Smith, C., Woolsey, E.S., Bourne, D.G., Bongaerts, P., Hoegh-Guldberg, O., and Leggat, W. (2015). The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts.&nbsp;ISME J&nbsp;9,&nbsp;2261-2274.</p>
<p>Apprill, A., Mcnally, S.P., Parsons, R., and Weber, L. (2015). Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton.&nbsp;Aquatic Microbial Ecology&nbsp;75,&nbsp;129-137.</p>
<p>Apprill, A., and Rappé, M.S. (2011).&nbsp;Response&nbsp;of the microbial community to coral spawning in lagoon and reef flat environments of Hawaii, USA.&nbsp;Aquatic Microbial Ecology&nbsp;62,&nbsp;251-266.</p>
<p>Budd, A.F., Fukami, H., Smith, N.D., and Knowlton, N. (2012). Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia).&nbsp;Zoological Journal of the Linnean Society&nbsp;166,&nbsp;465-529.</p>
<p>Holmes, R.M., Aminot, A., Kerouel, R., Hooker, B.A., and Peterson, B.J. (1999). A simple and precise method for measuring ammonium in marine and freshwater ecosystems.&nbsp;Canadian Journal of Fisheries and Aquatic Science,&nbsp;1801-1808.</p>
<p>Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S., and Schloss, P.D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.&nbsp;Applied and Environmental Microbiology&nbsp;79,&nbsp;5112-5120.</p>
<p>Parada, A., Needham, D.M., and Fuhrman, J.A. (2015). Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities,&nbsp;time-series&nbsp;and global field samples.&nbsp;Environmental Microbiology,&nbsp;1403-1414.</p>
<p>Taylor, B.W., Keep, C.F., Hall, R.O., Koch, B.J., Tronstad, L.M., Flecker, A.S., and Ulseth, A.J. (2007). Improving the fluorometric ammonium method: matrix effects, background fluorescence, and standard additions.&nbsp;Journal of The North American Benthological Society&nbsp;26,&nbsp;167-177.</p>
<p>Van Heukelem, L., and Thomas, C.S. (2001). Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis&nbsp;ofphytoplankton&nbsp;pigments.&nbsp;Journal of Chromatography&nbsp;910,&nbsp;31-49.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1233612 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1233612
completed
Amy Apprill
Woods Hole Oceanographic Institution
508-289-2649
Marine Chemistry and Geochemistry Department 266 Woods Hole Road, MS# 4
Woods Hole
MA
02543
USA
aapprill@whoi.edu
pointOfContact
asNeeded
Dataset Version: Final
Unknown
colony
species
site
lat
lon
depth
tissue_accessionNumber
tissue_accessionLink
mucus_accessionNumber
mucus_accessionLink
holobiont_accessionNumber
holobiont_accessionLink
filename1
filename2
filename3
Airbrush with 80 psi air pressure
MiSeq
Centrifuge
Continous Segmented Flow System
Bio-Rad thermocycler
EXO Water Quality Sonde
theme
None, User defined
sample identification
species scientific name (binomial)
site
latitude
longitude
depth
accession number
file_link
featureType
BCO-DMO Standard Parameters
Airbrush
Automated DNA Sequencer
Centrifuge
Continuous Flow Analyzer
Thermal Cycler
Water Quality Multiprobe
instrument
BCO-DMO Standard Instruments
Apprill_2013
service
Deployment Activity
Bermuda, Red Sea, Federated States of Micronesia, Florida Keys, and Australia
place
Locations
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Fundamental Coral-Microbial Associations
https://osprey.bco-dmo.org/project/564442
Fundamental Coral-Microbial Associations
<p><em>Description from NSF award abstract:</em><br />
Reef-building corals are in decline worldwide due in part to climate change and other human activities, and it is becoming increasingly important to understand what aspects of coral biology are degraded by environmental stress which then leads to coral mortality. It is now widely known that corals harbor communities of bacteria and archaea that are believed to play important roles in maintaining the health of their hosts, but we lack any appreciable understanding about the identity of the microbial associates regularly residing within healthy, reef-building corals. This project asks the central question: do reef-building corals harbor fundamental or persistent microbial associates that are symbiotic within their tissues? In order to address this hypothesis, the investigator will assess the identity of the bacterial and archaeal microbes using a variety of molecular and microscopy approaches that includes the identification and localization of a widespread group of coral bacterial associates belonging to the genus Endozoicomonas. The results of this study will then be used to develop additional questions about the role of these microbial associates in nutrient cycling and how they contribute to the health and survival of corals.</p>
Coral Microbial Relationships
largerWorkCitation
project
eng; USA
oceans
Bermuda, Red Sea, Federated States of Micronesia, Florida Keys, and Australia
2016-10-19
Florida Keys, Federated States of Micronesia, Red Sea, & Bermuda
0
BCO-DMO catalogue of parameters from Accession numbers and images of coral samples taken in the Florida Keys during 2013 (Coral Microbial Relationships project)
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/662074.rdf
Name: colony
Units: unitless
Description: <p>PI issued colony ID</p>
http://lod.bco-dmo.org/id/dataset-parameter/662075.rdf
Name: species
Units: unitless
Description: <p>Coral species sampled</p>
http://lod.bco-dmo.org/id/dataset-parameter/662076.rdf
Name: site
Units: unitless
Description: <p>Site where sampling took place</p>
http://lod.bco-dmo.org/id/dataset-parameter/662077.rdf
Name: lat
Units: decimal degrees
Description: <p>Latitude; North is positive</p>
http://lod.bco-dmo.org/id/dataset-parameter/662078.rdf
Name: lon
Units: decimal degrees
Description: <p>Longitude; West is positive</p>
http://lod.bco-dmo.org/id/dataset-parameter/662079.rdf
Name: depth
Units: meters
Description: <p>Depth where sampling took place</p>
http://lod.bco-dmo.org/id/dataset-parameter/662080.rdf
Name: tissue_accessionNumber
Units: unitless
Description: <p>Tissue sample GenBank accession number (16S rRNA gene)</p>
http://lod.bco-dmo.org/id/dataset-parameter/662081.rdf
Name: tissue_accessionLink
Units: unitless
Description: <p>Tissue sample GenBank accession number link</p>
http://lod.bco-dmo.org/id/dataset-parameter/662082.rdf
Name: mucus_accessionNumber
Units: unitless
Description: <p>Mucus sample GenBank accession number (16S rRNA gene)</p>
http://lod.bco-dmo.org/id/dataset-parameter/662083.rdf
Name: mucus_accessionLink
Units: unitless
Description: <p>Mucus sample GenBank accession number link</p>
http://lod.bco-dmo.org/id/dataset-parameter/662084.rdf
Name: holobiont_accessionNumber
Units: unitless
Description: <p>Holobiont sample GenBank accession number (16S rRNA gene)</p>
http://lod.bco-dmo.org/id/dataset-parameter/662085.rdf
Name: holobiont_accessionLink
Units: unitless
Description: <p>Holobiont sample GenBank accession link</p>
http://lod.bco-dmo.org/id/dataset-parameter/662086.rdf
Name: filename1
Units: unitless
Description: <p>First image of coral colony sampled</p>
http://lod.bco-dmo.org/id/dataset-parameter/662087.rdf
Name: filename2
Units: unitless
Description: <p>Second image of coral colony sampled</p>
http://lod.bco-dmo.org/id/dataset-parameter/662088.rdf
Name: filename3
Units: unitless
Description: <p>Third image of coral colony sampled</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
42441
https://datadocs.bco-dmo.org/file/zqqADPktnknm9x/coral_images.csv
coral_images.csv
Primary data file for dataset ID 661973
download
https://osprey.bco-dmo.org/dataset/661973/data/download
download
onLine
dataset
<p><strong>Sample collections:&nbsp;</strong></p>
<p>Triplicate colonies of the corals&nbsp;Orbicella&nbsp;(formerly&nbsp;Montastrea)&nbsp;faveolata&nbsp;(Budd et al., 2012),&nbsp;Montastrea&nbsp;cavernosa,&nbsp;Diploria&nbsp;strigosa,&nbsp;Porites&nbsp;astreoides&nbsp;and&nbsp;Porites porites&nbsp;were sampled via SCUBA diving using hammer and chisel at four sites within the Florida Keys, offshore of Summerland Key at depths ranging from 2.4 – 7.6 m (Reef flat, 24 deg 33.155’, 81 deg 22.88’; Open water patch reef, 24 deg 33.164, 81 deg 26.225; Mid-channel patch reef 24 deg 33.620, 81 deg 30.076; Nearshore reef, 24 deg 36.341, 81 deg 25.756 and an offshore Nursery site, 24 deg 33.745, 81 deg 24.013, where corals had been transplanted from nearby reefs). Fragments from each colony were placed in sterile Whirl-Pak bags (Nasco, Fort Atkinson WI, USA) underwater and transferred to a cooler containing ice after dive completion.&nbsp; Within 1-4 hours, each fragment was rinsed with 0.1 um filtered seawater. Coral mucus was collected by siphoning the mucus from the coral surface with a pipette, and mucus fractions were frozen at -80 deg C.&nbsp; The coral fragment was then divided using a sterilized chisel and hammer into two smaller fragments; one frozen at -80 deg C and the second placed in a 4% paraformaldehyde-0.2 um filter sterilized phosphate buffered saline solution, fixed at 4 deg C for 1 hour, and stored at -20 deg C.</p>
<p>At each site, seawater temperature, dissolved oxygen, and pH were measured&nbsp;from&nbsp;a depth just above the corals (~5 m) and from the surface (0-1 m) using an EXO water quality sonde (YSI Inc., Yellow Springs, OH, USA).&nbsp; Seawater samples (4 l) from the same depths were also collected for microbial biomass, inorganic nutrient&nbsp;analyses&nbsp;and direct cell enumeration, and stored on ice for 1-4 hours.&nbsp; Upon arrival at the laboratory, water for nutrient analyses was frozen at -20 deg C.&nbsp; For direct cell counts, duplicate 1 ml aliquots of seawater were preserved at a final concentration of 4% paraformaldehyde for 20 minutes at 4 deg C and then frozen at -80 deg C. &nbsp; Finally, ~2 l of water was pressure filtered onto duplicate 25 mm, 0.22 um Durapore membrane filters (Millipore, Boston, MA, USA) using a peristaltic pump for&nbsp;collection&nbsp;of seawater microbial biomass and stored at -80 deg C.</p>
<p><strong>Nutrient and pigment analyses and direct cell&nbsp;counts:&nbsp;</strong></p>
<p>Concentrations of dissolved inorganic nutrients (NH4+, NO3–&nbsp;+ NO2–, NO2-, PO43–, and silicate) were measured using a continuous segmented flow system with methods previously described (Apprill and Rappé, 2011), with NO3-&nbsp;was derived from subtracting the contribution of NO2-.&nbsp;&nbsp;NH4+&nbsp;was also measured in the field on unfrozen samples using the ortho-phthaldialdehyde&nbsp;fluorescence method (Holmes et al., 1999;Taylor et al., 2007).&nbsp; Pigment analysis of the phytoplankton community was performed using&nbsp;high-performance&nbsp;liquid chromatography with known standards on extracts from the frozen filters, and quantified as described previously (Van Heukelem and Thomas, 2001). Seawater microbial cell counts were measured using flow cytometry (Apprill and Rappé, 2011). &nbsp;</p>
<p><strong>Preparation of nucleic acids:</strong>&nbsp;</p>
<p>In order to examine mucus- and tissue-associated microbes, coral samples were processed using three approaches (Fig. 1A). &nbsp; The first approach harvested the mucus that was collected as previously described (hereafter referred to as ‘mucus’ samples). For the second approach, the frozen coral tissue (which still contained some mucus) was removed from the skeleton using an airbrush with 80 psi air pressure and 0.22 um filtered phosphate buffered saline solution.&nbsp; The tissue homogenate was then vortexed for 2 min and centrifuged at 20,000 x g for 20 min at 4 deg C. After removal of the supernatant, the cells were stored at -80 deg C for 1 week or less (referred to as ‘holobiont’ samples).&nbsp; Lastly, the third biomass substrate analyzed was decalcified coral tissue, referred to as ‘tissue’ samples, which were devoid of mucus and skeleton. To obtain these samples, the coral subsample that was initially preserved in paraformaldehyde solution was placed in a 20% EDTA solution (Acros Organics Thermo Fisher Scientific, NJ, USA) for 2-3 weeks at 4C on a slow rocker. The EDTA solution was exchanged daily until complete skeleton dissolution, (similar to Ainsworth et al., 2015).&nbsp;</p>
<p>Nucleic acids were extracted from the seawater membrane filters (1/4 of the 25 mm filter), the&nbsp;holobiont&nbsp;cells, and the decalcified tissue samples using the PowerPlant Pro DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA), with modifications that included adaptations that included 350 mg of 0.1mm silica beads (MP Biomedicals, Irvine, CA, USA) to the bead solution and 25 ul of proteinase K (Qiagen Inc., Valencia, CA, USA) followed by incubation at 65 deg C for 60 min. Because tissue samples were initially preserved in paraformaldehyde, they were subject to an extended&nbsp;30-minute&nbsp;proteinase K digestion at 55 deg C followed by an additional high-heat incubation step at 90 deg C for 60 minutes before&nbsp;beadbeating. The PowerPlant Pro extraction method did not result in&nbsp;high-quality&nbsp;DNA from the mucus.&nbsp; Therefore, the PowerBiofilm DNA Isolation kit (Mo Bio Laboratories), containing inhibitor removal steps but otherwise a similar bead mixture and proteinase K digestion at 65 deg C for 60 min, was used for mucus samples. All nucleic acids were quantified using the Qubit dsDNA BR fluorescent assay (Invitrogen Corp., Carlsbad, CA, USA) on a Qubit 2.0 fluorometer.</p>
<p><strong>Sequencing of&nbsp;V4&nbsp;region of archaeal and bacterial SSU rRNA genes:&nbsp;</strong></p>
<p>Barcoded primers targeting the V4 hypervariable region of the SSU rRNA gene,&nbsp;515F&nbsp;and 806R, were utilized for sequencing analysis as detailed in&nbsp;Kozich&nbsp;and colleagues (2013).&nbsp; This sequencing and data analysis occurred prior to the modifications of these primers to capture additional SAR11 clade bacteria (Apprill et al., 2015) and Thaumarchaeota (Parada et al., 2015), and therefore SAR11 are likely underrepresented by 15-25% in the seawater samples; Thaumarchaeota are probably not heavily underestimated in this study because the archaeal&nbsp;amoA&nbsp;gene abundances suggest their low abundance in reef seawater.&nbsp; Triplicate 25 ul PCR reactions contained 1.25 U of GoTaq Flexi DNA Polymerase (Promega Cooperation, Madison, WI, U.S.A.), 5X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, 200 uM of each dNTP, 200 nM of each barcoded primer, and 0.15 ng - 2.0 ng of genomic template for most samples, with some samples diluted or concentrated to encompass a range of 0.0057 to 4.40 ng. The reaction conditions included an initial denaturation step at 95 ºC for 2 min, followed by 32-39 cycles of 95 ºC for 20 s, 55 ºC for 15 s, and 72 ºC for 5 min, concluding with an extension at 72 ºC for 10 min. The reactions were carried out on a thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction products (5 ul) were screened on a 1% agarose-TBE gel.&nbsp; Samples were optimized for PCR at the lowest number of cycles that resulted in an amplified PCR product detected on a gel with the HyperLadder II standard (generally 5 ng ul-1) (Bioline, Taunton, MA, USA), thus minimizing biases from over-amplification.&nbsp; The three replicate reactions were excised from the gel, combined, and purified using the Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, California, USA), and quantified using the Qubit dsDNA HS fluorescent assay.&nbsp; Barcoded amplicons were pooled in equimolar ratios (5 ng each) and shipped to the University of Illinois W.M. Keck Center for Comparative and Functional Genomics for&nbsp;construction&nbsp;of libraries and sequenced using 2x250 bp paired-end MiSeq (Illumina, San Diego, CA, USA).</p>
<p><strong>References:</strong></p>
<p>Ainsworth, T.D., Krause, L., Bridge, T., Torda, G., Raina, J.-B., Zakrzewski, M., Gates, R.D., Padilla-Gamino, J.L., Spalding, H.L., Smith, C., Woolsey, E.S., Bourne, D.G., Bongaerts, P., Hoegh-Guldberg, O., and Leggat, W. (2015). The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts.&nbsp;ISME J&nbsp;9,&nbsp;2261-2274.</p>
<p>Apprill, A., Mcnally, S.P., Parsons, R., and Weber, L. (2015). Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton.&nbsp;Aquatic Microbial Ecology&nbsp;75,&nbsp;129-137.</p>
<p>Apprill, A., and Rappé, M.S. (2011).&nbsp;Response&nbsp;of the microbial community to coral spawning in lagoon and reef flat environments of Hawaii, USA.&nbsp;Aquatic Microbial Ecology&nbsp;62,&nbsp;251-266.</p>
<p>Budd, A.F., Fukami, H., Smith, N.D., and Knowlton, N. (2012). Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia).&nbsp;Zoological Journal of the Linnean Society&nbsp;166,&nbsp;465-529.</p>
<p>Holmes, R.M., Aminot, A., Kerouel, R., Hooker, B.A., and Peterson, B.J. (1999). A simple and precise method for measuring ammonium in marine and freshwater ecosystems.&nbsp;Canadian Journal of Fisheries and Aquatic Science,&nbsp;1801-1808.</p>
<p>Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S., and Schloss, P.D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.&nbsp;Applied and Environmental Microbiology&nbsp;79,&nbsp;5112-5120.</p>
<p>Parada, A., Needham, D.M., and Fuhrman, J.A. (2015). Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities,&nbsp;time-series&nbsp;and global field samples.&nbsp;Environmental Microbiology,&nbsp;1403-1414.</p>
<p>Taylor, B.W., Keep, C.F., Hall, R.O., Koch, B.J., Tronstad, L.M., Flecker, A.S., and Ulseth, A.J. (2007). Improving the fluorometric ammonium method: matrix effects, background fluorescence, and standard additions.&nbsp;Journal of The North American Benthological Society&nbsp;26,&nbsp;167-177.</p>
<p>Van Heukelem, L., and Thomas, C.S. (2001). Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis&nbsp;ofphytoplankton&nbsp;pigments.&nbsp;Journal of Chromatography&nbsp;910,&nbsp;31-49.</p>
Specified by the Principal Investigator(s)
<p><strong>BCO-DMO Data Processing Notes:</strong></p>
<p>-added accession links column for each accession number listed<br />
-removed spaces and replaced with underscores<br />
-filled in all blank cells with nd<br />
-reformatted column names to comply with BCO-DMO standards<br />
-added image links to data<br />
-converted all lat/lons to decimal degrees</p>
Specified by the Principal Investigator(s)
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
Airbrush with 80 psi air pressure
Airbrush with 80 psi air pressure
PI Supplied Instrument Name: Airbrush with 80 psi air pressure PI Supplied Instrument Description:Frozen coral tissue was removed from skeleton using airbrush. Instrument Name: Airbrush Instrument Short Name:Airbrush Instrument Description: Device for spraying liquid by means of compressed air.
MiSeq
MiSeq
PI Supplied Instrument Name: MiSeq PI Supplied Instrument Description:MiSeq (Illumina, San Diego, CA, USA) Instrument Name: Automated DNA Sequencer Instrument Short Name:Automated Sequencer Instrument Description: A DNA sequencer is an instrument that determines the order of deoxynucleotides in deoxyribonucleic acid sequences.
Centrifuge
Centrifuge
PI Supplied Instrument Name: Centrifuge PI Supplied Instrument Description:Tissue homogenate was then vortexed for 2 min and centrifuged at 20,000 x g for 20 min at 4 deg C Instrument Name: Centrifuge Instrument Short Name: Instrument Description: A machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities (e.g., cream from milk) or liquids from solids.
Continous Segmented Flow System
Continous Segmented Flow System
PI Supplied Instrument Name: Continous Segmented Flow System PI Supplied Instrument Description:Dissolved inorganic nutrients measured. Instrument Name: Continuous Flow Analyzer Instrument Short Name:CFA Instrument Description: A sample is injected into a flowing carrier solution passing rapidly through small-bore tubing.
Bio-Rad thermocycler
Bio-Rad thermocycler
PI Supplied Instrument Name: Bio-Rad thermocycler PI Supplied Instrument Description:Reactions carried out on a Bio-Rad thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Instrument Name: Thermal Cycler Instrument Short Name:Thermal Cycler Instrument Description: A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)
EXO Water Quality Sonde
EXO Water Quality Sonde
PI Supplied Instrument Name: EXO Water Quality Sonde PI Supplied Instrument Description:Temperature, DO, and pH measured just above the coral colony and at surface. Instrument Name: Water Quality Multiprobe Instrument Short Name:Water Quality Multiprobe Instrument Description: An instrument which measures multiple water quality parameters based on the sensor configuration.
Deployment: Apprill_2013
Apprill_2013
BIOS
BIOS
laboratory
Apprill_2013
Amy Apprill
Woods Hole Oceanographic Institution
BIOS
BIOS
laboratory