http://lod.bco-dmo.org/id/dataset/880711
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
2022-09-20
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Coral fragment surface area calculations utilizing two methods (tin foil and Image J) and corresponding zooxanthellae count data
2022-09-21
publication
2022-09-21
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2022-10-03
publication
https://doi.org/10.26008/1912/bco-dmo.880711.1
Cara L. Fiore
Appalachian State University
principalInvestigator
Amy Apprill
Woods Hole Oceanographic Institution
principalInvestigator
Cole G. Easson
Middle Tennessee State University
principalInvestigator
Christopher J. Freeman
College of Charleston
principalInvestigator
Alicia M. Reigel
Appalachian State University
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: Reigel, A. M., Easson, C. G., Apprill, A., Freeman, C. J., Bartley, M. M., Fiore, C. L. (2022) Coral fragment surface area calculations utilizing two methods (tin foil and Image J) and corresponding zooxanthellae count data. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2022-09-21 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.880711.1 [access date]
Coral Surface Area and Zooxanthellae Counts Dataset Description: Methods and Sampling: <p><strong>Sample Collection and Maintenance</strong><br />
Three coral species (one octocoral and two scleractinians) were utilized in this experiment. The octocoral, <em>Eunicea flexuosa</em>, was collected from Wonderland Reef (24.558694, -81.503528) within the Florida Keys National Marine Sanctuary (FKNMS) under FL saltwater fishing permit (Permit #: I-H1R76333834 held by A.M. Reigel). Three axial branch tips were clipped from each of 10 healthy <em>E. flexuosa</em> colonies located at depths of ~5-8 meters. Branch tips were kept in seawater and immediately transported to the outdoor land-based nursery (CAOS) at Mote Marine Laboratory at Summerland Key, FL where they were placed into a shaded, temperature- and pH-maintained flow-through tank and allowed to acclimate for ~24 hours. The two hard coral species, <em>Acropora cervicornis</em> and <em>Orbicella faveolata</em> were provided by Mote Marine Laboratory's field- (<em>A. cervicornis; </em>Coordinates: 24.562747, -81.400455) and land-based (<em>O. faveolata</em>) nurseries as permitted under the FKNMS-2015-163-A3. All hard coral fragments were placed in the same flow-through CAOS tanks as the <em>E. Flexuosa</em> samples and allowed to acclimate for ~24 hours.</p>
<p>To develop a representative sponge community for the Florida Keys reefs, we collected 5 individuals of each of 6 sponge species (<em>Niphates digitalis</em>, <em>Verongula rigida</em>, <em>Aplysina fulva</em>, <em>Aplysina cauliformis</em>, <em>Xestospongia muta</em>, <em>Callyspongia aculeata</em>) from Wonderland Reef under FL saltwater fishing permit (Permit #: I-H1R76333834 held by A.M. Reigel). Sponges were kept in seawater and immediately transported to the lab where they were placed in a shaded CAOS flow-through tank to acclimate for ~24 hours. Corals and sponges were not in the same CAOS tanks during the acclimation period.</p>
<p><strong>Coral and Zooxanthellae Separations</strong><br />
To prepare for downstream analyses the coral fractions, host and zooxanthellae, were manually separated. Scleractinian fragments were thawed and airbrushed with an aerosolized jet of 0.22 um filtered seawater to physically separate the coral tissue and skeleton and suspend the coral tissue material into a homogenate. To separate host tissue from zooxanthellae cells, the homogenate was centrifuged at 2000g for 3-5 minutes. Centrifugation formed a pellet comprised of zooxanthellae cells and a homogenate of host material. The host homogenate was pipetted into a separate sterile 50 ml Falcon tube. The homogenate, zooxanthellae pellet, and skeletal fragments were frozen and transported to Appalachian State University where they were stored at -20F until further processing. At Appalachian State, host homogenates and zooxanthellae pellets were thawed and checked for purity. Impure fractions were combined, homogenized with a tissue homogenizer (maximum speed for ~15 sec) to physically separate zooxanthellae cells from host tissue, and centrifuged (3000 x g, 6 min.) to pellet the zooxanthellae cells. Separated fractions were combined with original fractions each time and checked for purity under the microscope. The process was repeated until at least 80% purity was reached.</p>
<p><em>E. flexuosa</em> fractions were separated using a different process. First, the frozen coral branches were lyophilized (Labconco™ FreeZone™ Bulk Tray Dryer) for 22-24 hours, until they were completely dry. Following lyophilization, the axial skeleton was removed and the tissue was ground up using a mortar and pestle (note: separate mortar and pestle sets were used for control and enriched samples). The ground tissue was weighed and then rehydrated in 10ml of MilliQ water in a sterile 15ml Falcon tube. Very quickly following rehydration, the sclerites (skeletal fragments) sank to the bottom of the tube and the remaining host homogenate was pipetted into a new tube taking care not to transfer the sclerites. The homogenate was homogenized using a tissue homogenizer for ~15 seconds at maximum speed and centrifuged at 4000g for 5 minutes to separate the fractions. The centrifugation step was repeated as necessary until the host homogenate and zooxanthellae pellets were pure. Following both octocoral and scleractinian fraction separations, 50 ul of pure zooxanthellae from each sample was transferred to a cryovial with 50 ul of 10% paraformaldehyde (PFA) to fix the cells and stored in the refrigerator for future zooxanthellae counts. The pure host homogenates and remaining zooxanthellae pellets were stored in the -20F freezer.</p>
<p><strong>Coral Surface Area Measurements</strong><br />
Coral fragment surface area was calculated following two well-documented methods: Image J measurements and the aluminum foil method (Marsh 1970). ImageJ coral surface area measurements were completed utilizing planar photography and ImageJ software (Schneider et al. 2012). For <em>A. cervicornis</em> and <em>O. faveolata</em>, fragment surface area was measured using the frozen, airbrushed skeleton for each sample, while for <em>E. flexuosa</em>, the entire branch, prior to lyophilization as detailed above, was used. To obtain images of <em>A. cervicornis</em> and <em>E. flexuosa</em>, the fragments were held at an upright position, similar to their natural growth direction, and photographed from four sides (rotated 90°). <em>O. faveolata</em> are dome-shaped mounding corals, so photographs were only taken from above. A ruler was held in alignment with the fragments for scaling purposes. Photographs were individually uploaded to ImageJ and pixel dimensions were set using the straight-line tool and ‘set scale’ option. Using the polygon tool to drag an outline around the perimeter of the fragment, the enclosed area was calculated with the ‘measure’ function (in cm²). The area of all four sides was summed to estimate the surface area of the skeletal fragment.</p>
<p>The aluminum foil method was completed as documented in Marsh (1970), but briefly, small pieces of aluminum foil were cut and carefully measured (cm²) and weighed (g) to obtain a standard weight per unit of area (g/cm²) for the foil. The foil used in this study had a standard weight per unit of area of 0.00618 g/cm². Coral skeletal fragments or octocoral branches were carefully covered with aluminum foil and all excess foil was trimmed until there was no overlap. Each foil wrapping was carefully removed from the fragment and weighed. The fragment surface area was calculated using the standard weight per unit area of the aluminum foil (Surface area of coral fragment = mass of coral fragment foil*0.006180525794 g/cm²). The surface area calculations from both methods were compared for all fragments to ensure a relative consensus between the methods. Results were largely similar between methods and we chose to utilize the surface area estimates from the ImageJ method for downstream analyses as it resulted in slightly higher surface area values, which we deemed more conservative.</p>
<p><strong>Zooxanthellae Counts</strong><br />
To obtain counts of zooxanthellae cells per coral fragment, 20 ul of the fixed zooxanthellae cells from each sample were stained with either 2 ul (<em>E. flexuosa</em>) or 5 ul (<em>A. cervicornis</em> and <em>O. faveolata</em>) of Trypan Blue to increase their visibility under the microscope. If the zooxanthellae cells were too numerous to be counted accurately, they were further diluted with 175 ul of MilliQ water. 10 ul of the dilution was then placed into the cell counting chamber of a hemocytometer (Marienfeld Superior Neubauer Improved Chamber) and cells were counted in 4 quadrants following the standard Neubauer protocol as suggested by Electron Microscopy Sciences (<a href="https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx" target="_blank">https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx</a>). We used the following calculations to obtain whole fragment zooxanthellae counts and densities for each coral sample:</p>
<p><strong>Zooxanthellae cells/ml of dilution</strong> = (Zooxanthellae cell count*Dilution Factor*10,000 cells/ml)/4 quadrants</p>
<p><strong>Total zooxanthellae cells/fragment</strong> = Zooxanthellae cells/ml of dilution*Total Volume of host homogenate</p>
<p><strong>Zooxanthellae cells/cm</strong>²<strong> of coral fragment</strong> = Total zooxanthellae cells/fragment*fragment surface area (cm²)</p>
<p><strong>Known Issues/Problems</strong><br />
Three coral samples (Ef-iso-T3-7, Ef-iso-T6-2, Ac-iso-I-2) were not utilized for zooxanthellae counts because either no zooxanthellae cells were seen in the sample or, during the host and symbiont separations, the ethanol cleaning step was not fully rinsed, which causes the zooxanthellae to burst and therefore they are unable to be counted. These details are noted in the "Notes" column of the dataset.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1924540 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1924540
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1923962 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1923962
completed
Cara L. Fiore
Appalachian State University
828-262-7548
572 Rivers St
Boone
NC
28607
USA
fiorec@appstate.edu
pointOfContact
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
Cole G. Easson
Middle Tennessee State University
615-898-5822
1672 Greenland Drive SCI 2044
Murfreesboro
TN
37132
USA
Cole.Easson@mtsu.edu
pointOfContact
Christopher J. Freeman
College of Charleston
817-707-5840
66 George Street Department of Biology
Charleston
SC
29424
USA
freemancj@cofc.edu
pointOfContact
Alicia M. Reigel
Appalachian State University
Lexington
VA
USA
areigel@wlu.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Sample_ID
Coral_Species
TimePoint
Treatment
Field_Sampling_Date
Latitude
Longitude
Skeletal_fragment_diameter
Skeletal_fragment_length
ImageJ_side_1
ImageJ_side_2
ImageJ_side_3
ImageJ_side_4
ImageJ_total_surface_area
Mass_of_tin_foil
Foil_Fragment_Surface_Area
Zooxanthellae_Cell_Count
Quadrants_counted
Total_Volume_of_host_homogenate
Volume_of_fixed_zooxanthellae_homogenate_in_Dilution
Volume_of_Trypan_Added_to_Dilution
Volume_Of_MilliQ_Water_added_to_Dilution
Total_volume_added_to_dilution
Dilution_Factor
Zoox_cells_per_mL_dilution
Zoox_cells_per_fragment
Surface_area
Cells_per_cm2
Notes
Labconco™ FreeZone™ Bulk Tray Dryer
Marienfeld Superior Neubauer Improved Chamber
mortar and pestle
theme
None, User defined
sample identification
species scientific name (binomial)
time_point
treatment
date
latitude
longitude
diameter
length
surface_area
mass
count
volume
dilution
abundance
comments
featureType
BCO-DMO Standard Parameters
Centrifuge
Drying Oven
Hemocytometer
Homogenizer
Microscope - Optical
instrument
BCO-DMO Standard Instruments
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.
Collaborative Research: The Influence of Sponge Holobiont Metabolism on Coral Reef Dissolved Organic Matter and Reef Microorganisms
https://osprey.bco-dmo.org/project/822468
Collaborative Research: The Influence of Sponge Holobiont Metabolism on Coral Reef Dissolved Organic Matter and Reef Microorganisms
<p><em>NSF Award Abstract:</em><br />
The seawater around coral reefs is typically low in nutrients, yet coral reefs are teeming with life and are often compared to oases in a desert. Life exists in these 'marine deserts' in large part, due to symbiotic associations between single-celled microbes and invertebrates such as corals and sponges. The concentration and type of dissolved organic matter (DOM), a complex pool of organic nutrients such as amino acids, vitamins, and other diverse compounds, also affects the health of coral reefs. The composition of DOM on coral reefs is linked to both the composition of free-living microbes in the seawater and to the nutrition of filter-feeding organisms, such as corals and sponges. However, the factors that influence the composition of DOM on coral reefs and the consequences of how it changes are not well understood. Recent work suggests that sponges could have a significant impact on the composition of reef dissolved organic nutrients, depending on sponge species due to differences in filtration capacity and in their symbiotic microbial communities. This project characterizes how diverse sponge species process DOM on coral reefs and determines the impacts of this processing on the free-living microbial community. Seawater is collected from sponges (pre- and post- sponge filtration) on coral reefs in the relatively pristine region of Curacao, and incubation experiments measure the impact of sponge filtration on the growth of the free-living microbial community. The organic nutrients of seawater samples are analyzed using cutting-edge techniques to distinguish the types of nutrients that are processed by sponges. The incubation experiments, using free-living microbes collected from the coral reef, quantify the impact of sponge filtration on the growth and composition of this community. This project provides fundamental understanding of how sponges contribute to the base of the coral reef food web. As the human-driven impacts continue to alter the composition of organisms on reefs, this understanding is necessary to predict changes to reef microbial food webs and is thus essential for scientists, reef managers, and policy decision makers. This project trains undergraduate students and a postdoctoral scholar and contributes to undergraduate and K-12 education through development of sponge-centric lessons that focus on local U.S. east coast aquatic environments as well as coral reef ecosystems.</p>
<p>Sponges vary in their capacity to filter seawater and in their associated microbial communities, leading to diverse metabolic strategies that often coexist in one habitat. While it is well-established that sponges are important in processing dissolved organic matter (DOM), an important reservoir of reduced carbon compounds, and transferring this energy to benthic food webs, there has been limited work to understand the consequences of sponge processing on the composition of coral reef DOM and on pelagic food webs. Specifically, while studies have shown that exudates of corals and algae select for specific groups of picoplankton (autotrophic and heterotrophic, respectively), similar data for sponges are required to understand the multiple factors that shape the composition of DOM and of the picoplankton community on coral reefs. Thus, this project is aimed at addressing a major knowledge gap of the role of sponge-derived DOM (sponge exometabolome) in coral reef biogeochemistry. An in situ sampling design targeting prominent Caribbean sponges and picoplankton incubation experiments is coupled to address both the composition of sponge exometabolomes and delineate shifts in the picoplankton community derived from sponge exometabolomes. Molecular-level changes to seawater DOM by sponge processing and the impact of these changes on the overall coral reef DOM profile is assessed with two DOM analysis techniques: a commonly used fluorometry technique (fDOM analysis) and with high-resolution mass spectrometry (LC-MS/MS). Additionally, microbiome and functional gene profiling, growth metrics, and nutrient analyses are employed to assess changes in the picoplankton community in response to sponge exometabolomes. Advanced data analysis techniques then synthesize data generated by each approach to provide novel insight on a poorly uncharacterized biogeochemical pathway on coral reefs. The work outlined here represents entirely novel information on the impact of sponge metabolism on the composition of DOM, sheds light on biologically important molecules involved in benthic-pelagic coupling, and importantly, generates data using standardized methods, thus facilitating comparison to previous and future DOM datasets.</p>
<p>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.</p>
Sponge Holobiont DOM
largerWorkCitation
project
eng; USA
oceans
-81.503528
-81.400455
24.558694
24.562747
2020-12-09
2020-12-09
Caribbean Sea
0
BCO-DMO catalogue of parameters from Coral fragment surface area calculations utilizing two methods (tin foil and Image J) and corresponding zooxanthellae count data
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/880728.rdf
Name: Sample_ID
Units: unitless
Description: <p>Sample identifier</p>
http://lod.bco-dmo.org/id/dataset-parameter/880729.rdf
Name: Coral_Species
Units: unitless
Description: <p>Species name</p>
http://lod.bco-dmo.org/id/dataset-parameter/880730.rdf
Name: TimePoint
Units: unitless
Description: <p>Time point of the experiment. T0 = initial, T3= 3 hrs into the chase, T6 = 6 hrs into the chase</p>
http://lod.bco-dmo.org/id/dataset-parameter/880731.rdf
Name: Treatment
Units: unitless
Description: <p>Control or enriched with stable isotopes</p>
http://lod.bco-dmo.org/id/dataset-parameter/880732.rdf
Name: Field_Sampling_Date
Units: unitless
Description: <p>Field sampling date in format YYYY-MM-DD</p>
http://lod.bco-dmo.org/id/dataset-parameter/880733.rdf
Name: Latitude
Units: degrees North
Description: <p>Latitude of field sampling site</p>
http://lod.bco-dmo.org/id/dataset-parameter/880734.rdf
Name: Longitude
Units: degrees East
Description: <p>Longitude of field sampling site</p>
http://lod.bco-dmo.org/id/dataset-parameter/880735.rdf
Name: Skeletal_fragment_diameter
Units: centimeters (cm)
Description: <p>Skeletal fragment diameter</p>
http://lod.bco-dmo.org/id/dataset-parameter/880736.rdf
Name: Skeletal_fragment_length
Units: centimeters (cm)
Description: <p>Skeletal fragment length</p>
http://lod.bco-dmo.org/id/dataset-parameter/880737.rdf
Name: ImageJ_side_1
Units: square centimeters (cm^2)
Description: <p>Surface area of side 1 of coral fragment</p>
http://lod.bco-dmo.org/id/dataset-parameter/880738.rdf
Name: ImageJ_side_2
Units: square centimeters (cm^2)
Description: <p>Surface area of side 2 of coral fragment</p>
http://lod.bco-dmo.org/id/dataset-parameter/880739.rdf
Name: ImageJ_side_3
Units: square centimeters (cm^2)
Description: <p>Surface area of side 3 of coral fragment</p>
http://lod.bco-dmo.org/id/dataset-parameter/880740.rdf
Name: ImageJ_side_4
Units: square centimeters (cm^2)
Description: <p>Surface area of side 4 of coral fragment</p>
http://lod.bco-dmo.org/id/dataset-parameter/880741.rdf
Name: ImageJ_total_surface_area
Units: square centimeters (cm^2)
Description: <p>Combined surface area of sides 1-4 from ImageJ</p>
http://lod.bco-dmo.org/id/dataset-parameter/880742.rdf
Name: Mass_of_tin_foil
Units: grams (g)
Description: <p>Mass of tinfoil used to wrap skeletal fragment</p>
http://lod.bco-dmo.org/id/dataset-parameter/880743.rdf
Name: Foil_Fragment_Surface_Area
Units: square centimeters (cm^2)
Description: <p>Surface area of coral fragment as determined by the tin foil method. Weight of tinfoil/ standard weight per cm2 of tin foil (0.006180525794 g/cm2)</p>
http://lod.bco-dmo.org/id/dataset-parameter/880744.rdf
Name: Zooxanthellae_Cell_Count
Units: number of cells
Description: <p>Zooxanthellae cells count, counted using a hemocytometer</p>
http://lod.bco-dmo.org/id/dataset-parameter/880745.rdf
Name: Quadrants_counted
Units: number of grids
Description: <p>Number of grids counted on the hemocytometer</p>
http://lod.bco-dmo.org/id/dataset-parameter/880746.rdf
Name: Total_Volume_of_host_homogenate
Units: milliliters (mL)
Description: <p>Total volume of host homogenate</p>
http://lod.bco-dmo.org/id/dataset-parameter/880747.rdf
Name: Volume_of_fixed_zooxanthellae_homogenate_in_Dilution
Units: milliliters (mL)
Description: <p>Volume of fixed zooxanthellae homogenate in dilution</p>
http://lod.bco-dmo.org/id/dataset-parameter/880748.rdf
Name: Volume_of_Trypan_Added_to_Dilution
Units: milliliters (mL)
Description: <p>Volume of Trypan added to dilution</p>
http://lod.bco-dmo.org/id/dataset-parameter/880749.rdf
Name: Volume_Of_MilliQ_Water_added_to_Dilution
Units: milliliters (mL)
Description: <p>Volume of MilliQ water added to dilution</p>
http://lod.bco-dmo.org/id/dataset-parameter/880750.rdf
Name: Total_volume_added_to_dilution
Units: milliliters (mL)
Description: <p>Total volume (Trypan + MilliQ) added to dilution</p>
http://lod.bco-dmo.org/id/dataset-parameter/880751.rdf
Name: Dilution_Factor
Units: milliliters (mL)
Description: <p>Fixed zoox volume/Total vol added to dilution</p>
http://lod.bco-dmo.org/id/dataset-parameter/880752.rdf
Name: Zoox_cells_per_mL_dilution
Units: cells per milliliter (cells/mL)
Description: <p>(Zooxanthellae cell count*dilution factor*10000 cells/ml standard for hemocytometer)/Grids Counted</p>
http://lod.bco-dmo.org/id/dataset-parameter/880753.rdf
Name: Zoox_cells_per_fragment
Units: cells per milliliter (cells/mL)
Description: <p>Total zoo cells in host homogenate Zoox cells/ml*Total volume host homogenate</p>
http://lod.bco-dmo.org/id/dataset-parameter/880754.rdf
Name: Surface_area
Units: square centimeters (cm^2)
Description: <p>Total surface area from ImageJ</p>
http://lod.bco-dmo.org/id/dataset-parameter/880755.rdf
Name: Cells_per_cm2
Units: cells per square centimeter (cells/cm2)
Description: <p>Total zoox cells in host homogenate*surface area (ImageJ)</p>
http://lod.bco-dmo.org/id/dataset-parameter/880756.rdf
Name: Notes
Units: unitless
Description: <p>Additional notes from zooxanthellae counting</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
17591
https://darchive.mblwhoilibrary.org/bitstream/1912/29379/1/dataset-880711_coral-surface-area-and-zooxanthellae-counts__v1.tsv
download
https://doi.org/10.26008/1912/bco-dmo.880711.1
download
onLine
dataset
<p><strong>Sample Collection and Maintenance</strong><br />
Three coral species (one octocoral and two scleractinians) were utilized in this experiment. The octocoral, <em>Eunicea flexuosa</em>, was collected from Wonderland Reef (24.558694, -81.503528) within the Florida Keys National Marine Sanctuary (FKNMS) under FL saltwater fishing permit (Permit #: I-H1R76333834 held by A.M. Reigel). Three axial branch tips were clipped from each of 10 healthy <em>E. flexuosa</em> colonies located at depths of ~5-8 meters. Branch tips were kept in seawater and immediately transported to the outdoor land-based nursery (CAOS) at Mote Marine Laboratory at Summerland Key, FL where they were placed into a shaded, temperature- and pH-maintained flow-through tank and allowed to acclimate for ~24 hours. The two hard coral species, <em>Acropora cervicornis</em> and <em>Orbicella faveolata</em> were provided by Mote Marine Laboratory's field- (<em>A. cervicornis; </em>Coordinates: 24.562747, -81.400455) and land-based (<em>O. faveolata</em>) nurseries as permitted under the FKNMS-2015-163-A3. All hard coral fragments were placed in the same flow-through CAOS tanks as the <em>E. Flexuosa</em> samples and allowed to acclimate for ~24 hours.</p>
<p>To develop a representative sponge community for the Florida Keys reefs, we collected 5 individuals of each of 6 sponge species (<em>Niphates digitalis</em>, <em>Verongula rigida</em>, <em>Aplysina fulva</em>, <em>Aplysina cauliformis</em>, <em>Xestospongia muta</em>, <em>Callyspongia aculeata</em>) from Wonderland Reef under FL saltwater fishing permit (Permit #: I-H1R76333834 held by A.M. Reigel). Sponges were kept in seawater and immediately transported to the lab where they were placed in a shaded CAOS flow-through tank to acclimate for ~24 hours. Corals and sponges were not in the same CAOS tanks during the acclimation period.</p>
<p><strong>Coral and Zooxanthellae Separations</strong><br />
To prepare for downstream analyses the coral fractions, host and zooxanthellae, were manually separated. Scleractinian fragments were thawed and airbrushed with an aerosolized jet of 0.22 um filtered seawater to physically separate the coral tissue and skeleton and suspend the coral tissue material into a homogenate. To separate host tissue from zooxanthellae cells, the homogenate was centrifuged at 2000g for 3-5 minutes. Centrifugation formed a pellet comprised of zooxanthellae cells and a homogenate of host material. The host homogenate was pipetted into a separate sterile 50 ml Falcon tube. The homogenate, zooxanthellae pellet, and skeletal fragments were frozen and transported to Appalachian State University where they were stored at -20F until further processing. At Appalachian State, host homogenates and zooxanthellae pellets were thawed and checked for purity. Impure fractions were combined, homogenized with a tissue homogenizer (maximum speed for ~15 sec) to physically separate zooxanthellae cells from host tissue, and centrifuged (3000 x g, 6 min.) to pellet the zooxanthellae cells. Separated fractions were combined with original fractions each time and checked for purity under the microscope. The process was repeated until at least 80% purity was reached.</p>
<p><em>E. flexuosa</em> fractions were separated using a different process. First, the frozen coral branches were lyophilized (Labconco™ FreeZone™ Bulk Tray Dryer) for 22-24 hours, until they were completely dry. Following lyophilization, the axial skeleton was removed and the tissue was ground up using a mortar and pestle (note: separate mortar and pestle sets were used for control and enriched samples). The ground tissue was weighed and then rehydrated in 10ml of MilliQ water in a sterile 15ml Falcon tube. Very quickly following rehydration, the sclerites (skeletal fragments) sank to the bottom of the tube and the remaining host homogenate was pipetted into a new tube taking care not to transfer the sclerites. The homogenate was homogenized using a tissue homogenizer for ~15 seconds at maximum speed and centrifuged at 4000g for 5 minutes to separate the fractions. The centrifugation step was repeated as necessary until the host homogenate and zooxanthellae pellets were pure. Following both octocoral and scleractinian fraction separations, 50 ul of pure zooxanthellae from each sample was transferred to a cryovial with 50 ul of 10% paraformaldehyde (PFA) to fix the cells and stored in the refrigerator for future zooxanthellae counts. The pure host homogenates and remaining zooxanthellae pellets were stored in the -20F freezer.</p>
<p><strong>Coral Surface Area Measurements</strong><br />
Coral fragment surface area was calculated following two well-documented methods: Image J measurements and the aluminum foil method (Marsh 1970). ImageJ coral surface area measurements were completed utilizing planar photography and ImageJ software (Schneider et al. 2012). For <em>A. cervicornis</em> and <em>O. faveolata</em>, fragment surface area was measured using the frozen, airbrushed skeleton for each sample, while for <em>E. flexuosa</em>, the entire branch, prior to lyophilization as detailed above, was used. To obtain images of <em>A. cervicornis</em> and <em>E. flexuosa</em>, the fragments were held at an upright position, similar to their natural growth direction, and photographed from four sides (rotated 90°). <em>O. faveolata</em> are dome-shaped mounding corals, so photographs were only taken from above. A ruler was held in alignment with the fragments for scaling purposes. Photographs were individually uploaded to ImageJ and pixel dimensions were set using the straight-line tool and ‘set scale’ option. Using the polygon tool to drag an outline around the perimeter of the fragment, the enclosed area was calculated with the ‘measure’ function (in cm²). The area of all four sides was summed to estimate the surface area of the skeletal fragment.</p>
<p>The aluminum foil method was completed as documented in Marsh (1970), but briefly, small pieces of aluminum foil were cut and carefully measured (cm²) and weighed (g) to obtain a standard weight per unit of area (g/cm²) for the foil. The foil used in this study had a standard weight per unit of area of 0.00618 g/cm². Coral skeletal fragments or octocoral branches were carefully covered with aluminum foil and all excess foil was trimmed until there was no overlap. Each foil wrapping was carefully removed from the fragment and weighed. The fragment surface area was calculated using the standard weight per unit area of the aluminum foil (Surface area of coral fragment = mass of coral fragment foil*0.006180525794 g/cm²). The surface area calculations from both methods were compared for all fragments to ensure a relative consensus between the methods. Results were largely similar between methods and we chose to utilize the surface area estimates from the ImageJ method for downstream analyses as it resulted in slightly higher surface area values, which we deemed more conservative.</p>
<p><strong>Zooxanthellae Counts</strong><br />
To obtain counts of zooxanthellae cells per coral fragment, 20 ul of the fixed zooxanthellae cells from each sample were stained with either 2 ul (<em>E. flexuosa</em>) or 5 ul (<em>A. cervicornis</em> and <em>O. faveolata</em>) of Trypan Blue to increase their visibility under the microscope. If the zooxanthellae cells were too numerous to be counted accurately, they were further diluted with 175 ul of MilliQ water. 10 ul of the dilution was then placed into the cell counting chamber of a hemocytometer (Marienfeld Superior Neubauer Improved Chamber) and cells were counted in 4 quadrants following the standard Neubauer protocol as suggested by Electron Microscopy Sciences (<a href="https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx" target="_blank">https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx</a>). We used the following calculations to obtain whole fragment zooxanthellae counts and densities for each coral sample:</p>
<p><strong>Zooxanthellae cells/ml of dilution</strong> = (Zooxanthellae cell count*Dilution Factor*10,000 cells/ml)/4 quadrants</p>
<p><strong>Total zooxanthellae cells/fragment</strong> = Zooxanthellae cells/ml of dilution*Total Volume of host homogenate</p>
<p><strong>Zooxanthellae cells/cm</strong>²<strong> of coral fragment</strong> = Total zooxanthellae cells/fragment*fragment surface area (cm²)</p>
<p><strong>Known Issues/Problems</strong><br />
Three coral samples (Ef-iso-T3-7, Ef-iso-T6-2, Ac-iso-I-2) were not utilized for zooxanthellae counts because either no zooxanthellae cells were seen in the sample or, during the host and symbiont separations, the ethanol cleaning step was not fully rinsed, which causes the zooxanthellae to burst and therefore they are unable to be counted. These details are noted in the "Notes" column of the dataset.</p>
Specified by the Principal Investigator(s)
<p><strong>BCO-DMO Processing:</strong><br />
- replaced "NA" with "nd" as missing data value ("no data");<br />
- converted dates to format YYYY-MM-DD:<br />
- renamed fields to comply with BCO-DMO naming conventions;<br />
- created separate columns for Latitude and Longitude;<br />
- replaced commas with semi-colons in the Notes column.</p>
Specified by the Principal Investigator(s)
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PI Supplied Instrument Name: 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.
Labconco™ FreeZone™ Bulk Tray Dryer
Labconco™ FreeZone™ Bulk Tray Dryer
PI Supplied Instrument Name: Labconco™ FreeZone™ Bulk Tray Dryer Instrument Name: Drying Oven Instrument Short Name: Instrument Description: a heated chamber for drying
Marienfeld Superior Neubauer Improved Chamber
Marienfeld Superior Neubauer Improved Chamber
PI Supplied Instrument Name: Marienfeld Superior Neubauer Improved Chamber Instrument Name: Hemocytometer Instrument Short Name:Hemocytometer Instrument Description: A hemocytometer is a small glass chamber, resembling a thick microscope slide, used for determining the number of cells per unit volume of a suspension. Originally used for performing blood cell counts, a hemocytometer can be used to count a variety of cell types in the laboratory. Also spelled as "haemocytometer". Description from:
http://hlsweb.dmu.ac.uk/ahs/elearning/RITA/Haem1/Haem1.html.
mortar and pestle
mortar and pestle
PI Supplied Instrument Name: mortar and pestle Instrument Name: Homogenizer Instrument Short Name:Homogenizer Instrument Description: A homogenizer is a piece of laboratory equipment used for the homogenization of various types of material, such as tissue, plant, food, soil, and many others.
PI Supplied Instrument Name: Instrument Name: Microscope - Optical Instrument Short Name: Instrument Description: Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope". Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/