http://lod.bco-dmo.org/id/dataset/954470
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
2025-02-26
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Bulk isotope data from sponges collected in Summerland Key in Florida between July 27 - August 19, 2021
2025-02-26
publication
2025-02-26
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2025-05-07
publication
https://doi.org/10.26008/1912/bco-dmo.954470.1
Cole G. Easson
Middle Tennessee State University
principalInvestigator
Cara L. Fiore
Appalachian State University
principalInvestigator
Christopher J. Freeman
College of Charleston
principalInvestigator
Robert W. Thacker
Stony Brook 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: Freeman, C. J., Easson, C. G., Fiore, C. L., Thacker, R. W., Jones, H. (2025) Bulk isotope data from sponges collected in Summerland Key in Florida between July 27 - August 19, 2021. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2025-02-26 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.954470.1 [access date]
carribean sponges - bulk isotopes Dataset Description: Methods and Sampling: <p>Sample collection<strong><em>:</em></strong>&nbsp;Fieldwork was conducted at Mote Marine Laboratory in Summerland Key,&nbsp;FL, USA, between July 27 and August 19, 2021. Replicate individuals of the four sponge species&nbsp;<em>Aplysina cauliformis</em>,&nbsp;<em>Iotrochota birotulata</em>,&nbsp;<em>Niphates digitalis</em>, and&nbsp;<em>Xestospongia muta</em>&nbsp;were collected from a shallow (7 m depth) patch reef (24°33’ N, 81°24’ W) offshore of Summerland Key (24°39’ N, 81°26’ W) via dive knives (with the spongocoel intact for the barrel/tube sponges by cutting the sponge from the substrate) using SCUBA, and were placed into plastic bags filled with seawater before being brought to the surface. After collection, all sponges were kept submerged in ambient seawater in large, sealed plastic bags in an insulated cooler during transit back to the lab. Upon return to Mote Marine Laboratory, sponges were attached with cable ties to plastic window screens, and held in large, flow-through raceways for 24 h for acclimatization. At no point during this process were sponges exposed to air.&nbsp;</p>
<p>Only healthy sponges (sponges confirmed to be actively pumping via use of fluorescein dye, and without any signs of necrosis) were processed for and used in experiments. For each experiment (and each treatment in the autotrophy experiment), four large individuals of each of the rope sponges&nbsp;<em>I. birotulata</em>&nbsp;and&nbsp;<em>A. cauliformis</em>&nbsp;collected from the reef were partitioned into seven subsamples for sampling at discrete time points. Both&nbsp;<em>X. muta</em>&nbsp;and&nbsp;<em>N. digitalis</em>&nbsp;were only sampled at four timepoints during each experiment because they have barrel/tube morphologies that cannot be successfully partitioned and subsampled, requiring the sacrifice of an entire individual for each timepoint. Therefore, sixteen small individuals of each of these two species were collected from the reef for each experiment. Experimental replicates were defined as follows: a replicate of&nbsp;<em>X. muta</em>&nbsp;consisted of one individual attached to a plastic window screen, which was placed in an 8 L, clear food-grade container; a replicate of&nbsp;<em>N. digitalis</em>&nbsp;consisted of four small individuals attached to a plastic window screen, which was placed into a 17.9 L, clear food-grade container; and replicates of&nbsp;<em>A. cauliformis</em>&nbsp;and&nbsp;<em>I. birotulata</em>&nbsp;consisted of all seven subsamples from the same individual attached to a plastic window screen, which was placed into a 17.9 L, clear food-grade container. In total, there were four replicates of each species collected at each time point in each experiment, with the exception the DOM experiment, during which multiple individuals, including all individuals of&nbsp;<em>A. cauliformis</em>,<em>&nbsp;</em>developed necroses in experimental tanks after 24h. The effected individuals were eliminated, limiting replication to only two timepoints (24h and 72h) during the chase for all species. Due to necroses of all individuals of&nbsp;<em>A. cauliformis&nbsp;</em>after 24h, the DOM experiment was repeated for this species only, using three healthy replicates.</p>
<p>Pulse-chase experiments<strong><em>:</em></strong>&nbsp;Three separate “pulse-chase” experiments were carried out to track the uptake and recycling of labeled compounds from three different resource pools. The first resource pool, DOM, consisted of&nbsp;<sup>13</sup>C-labeled glucose (35 μM) and galactose (35 μM), and&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N-labeled cell-free amino acids (range from 39-790 nM) and urea (2.2 μM). The second resource pool, POM, consisted of&nbsp;<sup>15</sup>N- and&nbsp;<sup>13</sup>C-labeled&nbsp;<em>Synechococcus&nbsp;</em>spp. bacteria incubated for 36 h in seawater containing 1.18 μM NaH<sup>13</sup>CO₃, 3.67 μM&nbsp;<sup>15</sup>NH₄Cl, and 0.117 M Na<sup>15</sup>NO₃, and&nbsp;<em>Oceanicola batsensis&nbsp;</em>incubated in 0.55 μM galactose, 1.18 μM NaH<sup>13</sup>CO₃, 3.67 μM&nbsp;<sup>15</sup>NH₄Cl, and 0.117 μM Na<sup>15</sup>NO₃. Finally, the third resource pool of inorganic compounds consisted of Na<sup>15</sup>NO₃ (0.117 μM) and NaH<sup>13</sup>CO₃ (1.18 μM). Labeled bacterial cells were added to filtered seawater for the POM experiment at a final combined concentration of 4.65 x10<sup>5</sup>&nbsp;cells ml<sup>-1</sup>&nbsp;for photosynthetic cells and 1.01 x 10<sup>6</sup>&nbsp;for heterotrophic cells (measured at the Center for Aquatic Cytometry at Bigelow Laboratory for Ocean Sciences). Concentrations of labeled resources were chosen to approximate the concentrations of corresponding inorganic and organic sources of carbon and nitrogen found in Caribbean seawater as closely as possible, as well as to match concentrations used in previous studies. In all incubations, experimental water was 0.7 μm (GF/F) filtered prior to the addition of tracers or labeled bacteria. Sponges were “pulsed” for 3 h in water containing these isotopically enriched resources, and then sponges were held in unenriched, flowing seawater for a 72-h “chase” period. Experimental samples were collected during the pulse at t=0.5 h, and during the chase at final times of t=12 h, 48 h, and 75 h (minus the 48h sampling in the DOM experiment). Prior to the start of the pulse, one of the seven subsamples of&nbsp;<em>A. cauliformis</em>&nbsp;and&nbsp;<em>I. birotulata</em>, and four individuals each of&nbsp;<em>X. muta</em>&nbsp;and&nbsp;<em>N. digitalis</em>&nbsp;were sampled and processed to serve as t = 0 h (natural abundance) samples.</p>
<p>To start the pulse, each experimental replicate was added to a food grade container containing filtered seawater and labeled compounds or food bacteria, as well as a recirculating pump used to maintain flow. Replicates of&nbsp;<em>X. muta</em>were held in 8 L, clear food grade containers holding 6 L of filtered seawater, while replicates of&nbsp;<em>I. birotulata</em>,&nbsp;<em>A. cauliformis</em>, and&nbsp;<em>N. digitalis</em>&nbsp;were held in 17.9 L containers holding 15 L of filtered seawater. These containers were all held in a large 4.6 m-diameter outdoor tank with a shallow seawater bath to control temperature in experimental tanks holding sponges. At the end of the pulse, the water level in the outdoor tank was raised to flood experimental tanks with unlabeled seawater (supplied from the canal adjacent to Mote Marine Lab, drawn from 10 m, and filtered through sand to 40 μm) throughout the chase. Temperature measurements were taken at regular intervals (30 min), and an additional 25°C supply of well water, with salinity equal to that of seawater, was supplemented to maintain a tank temperature between 29-32°C. During the day for all experiments, irradiance measurements were taken every 30 min to monitor for deviation outside of the irradiance that sponges experience at depth, which was 800 to 1000 μmol photons m<sup>-2</sup>&nbsp;s<sup>-1</sup>&nbsp;measured at depth during cloudless daytime conditions at the collection site. One layer of standard shade cloth was sufficient to keep irradiance within the normal limits of what the sponges experience at depth, and adjustments during the experiment were only made when irradiance went below the 800-1000 μmol photons m<sup>-2</sup>&nbsp;s<sup>-1</sup>&nbsp;range under cloudy conditions. When irradiance decreased, we removed the layer of shade cloth to increase exposure of the sponges to ambient sunlight.</p>
<p>To assess the role of both autotrophic and heterotrophic symbionts in the assimilation of inorganic compounds, the autotrophy experiment included both light and dark treatments. For this reason, each large individual of&nbsp;<em>I. birotulata</em>&nbsp;and&nbsp;<em>A. cauliformis&nbsp;</em>was partitioned into 14 subsamples, and 32 small individuals each of&nbsp;<em>N. digitalis&nbsp;</em>and&nbsp;<em>X. muta</em>&nbsp;were collected for this experiment. Half of the subsamples or individuals of each species were randomly assigned to a dark treatment (covered in opaque plastic tarp with irradiance values from HOBO loggers that detected 0 Lux throughout the pulse), and the other half to a light treatment (exposure to ambient irradiance as in other experiments). Samples in the dark treatment were covered during the pulse but were exposed to ambient irradiance during the chase so that we could monitor nutrient retention and recycling under ambient (light-dark cycle) conditions.</p>
<p>Sample fixation<strong><em>:</em></strong>&nbsp;After sampling during pulse-chase experiments, sponges were rinsed with 0.7 μm-filtered, unenriched seawater and lightly blotted with a paper towel before weighing to the nearest 0.001 g to obtain a wet weight. Afterwards, ~2 mm cross sections were collected in duplicate using a razor blade and placed into 2 mL cryovials to be fixed for SEM and NanoSIMS analysis; the remaining bulk tissue was frozen at -20°C for isotope analysis. The 2 mm cross-sections of sponge tissue were fixed in&nbsp;2.5% (w/v) glutaraldehyde + 1% (w/v) paraformaldehyde in 1.5X&nbsp;PHEM buffer (1.5X PHEM (90 mM PIPES, 37.5 mM HEPES, 15 mM EGTA, 3 mM&nbsp;MgSO4.7H<sub>2</sub>O), and 9% (w/v) sucrose at pH 7.4) and stored at 4°C&nbsp;for 12h before being rinsed 3x with PHEM buffer containing no fixative. Finally, 0.1 mL of fixative solution (10% of the concentration in the original solution) was added back to each sample to prevent any fouling during long-term storage at&nbsp;4°C.&nbsp;After being taken out of storage,&nbsp;cross-sections of sponge tissue were rinsed once more in fixative-free 1.5X PHEM buffer, digested in 5% hydrofluoric acid to break down siliceous spicules within the sponge tissue (note this was carried out for all samples to be consistent, even though&nbsp;<em>A. cauliformis</em>&nbsp;lacks spicules), and rinsed twice and stored in 1.5X PHEM buffer.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1929293 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1929293
completed
Cole G. Easson
Middle Tennessee State University
615-898-5822
1672 Greenland Drive SCI 2044
Murfreesboro
TN
37132
USA
Cole.Easson@mtsu.edu
pointOfContact
Cara L. Fiore
Appalachian State University
828-262-7548
572 Rivers St
Boone
NC
28607
USA
fiorec@appstate.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
Robert W. Thacker
Stony Brook University
631-632-9949
West 5510 FRK MEL LIB
Stony Brook
NY
11794
USA
robert.thacker@stonybrook.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Experiment
time
replicate
species_abbreviation
Species_name
Collection_date
lat
lon
Linear_corr_d15N
wt_perc_N
Atm_perc_15N
Linear_corr_d13C
wt_perc_C
Atm_perc_13C
theme
None, User defined
experiment type
sample time
replicate
species scientific name (binomial)
date
latitude
longitude
d15N
Nitrogen
d13C
Elemental carbon (C)
featureType
BCO-DMO Standard Parameters
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: Investigations into microbially mediated ecological diversification in sponges
https://osprey.bco-dmo.org/project/772220
Collaborative Research: Investigations into microbially mediated ecological diversification in sponges
<p><em>NSF Award Abstract:</em><br />
Coral reefs represent a paradox because, despite their immense productivity and biodiversity, they are found in nutrient-poor habitats that are equivalent to "marine deserts." High biodiversity is often associated with a division of resources that allows many types of organisms to coexist with minimal competition. Indeed, unlike many other organisms on coral reefs, sponges are adapted to efficiently remove bacteria, phytoplankton, and dissolved organic matter from seawater by filter-feeding. Sponges are a dominant component of coral reefs worldwide and in the Caribbean, where their biomass exceeds that of reef-building corals. For almost a quarter century, the success of sponges in the Caribbean has been linked to their filter-feeding ability. However, recent work demonstrated that coexisting sponges on Caribbean reefs host unique communities of bacteria that might allow sponges to access multiple pools of nutrients that are not available to other organisms. In this project, the investigators will test the hypothesis that ecologically dominant sponge species in the Caribbean have unique metabolic strategies that are mediated by their associations with microbes that live within the sponge body. This research will combine manipulative field experiments with a novel combination of modern analytical tools to investigate both filter-feeding by sponge hosts and the metabolic pathways of their microbes. This work will advance our understanding of the ecological and evolutionary forces that have helped shape the species present on Caribbean coral reefs. Additionally, this project will support three early-career investigators and provide training opportunities for graduate and undergraduate students at Nova Southeastern University, Appalachian State University, Stony Brook University, and Smithsonian Marine Station. The investigators will also develop innovative outreach programs that expand existing platforms at their institutions to increase public engagement and scientific literacy.</p>
<p>Marine sponges have been widely successful in their expansion across ecological niches in the Caribbean, with biomass often exceeding that of reef-building corals and high species diversity. However, whether this success is linked to efficient heterotrophic filter-feeding on organic carbon in the water column or to their evolutionary investment in microbial symbionts is yet to be fully elucidated. Microbial symbionts expand the metabolic capabilities of host sponges, supplementing heterotrophic feeding with inorganic carbon and nitrogen, mediating the assimilation of dissolved organic matter, and facilitating recycling of host-derived nitrogen. Despite these benefits, microbial symbiont communities are widely divergent across coexisting sponge species and there is substantial variation in host reliance on symbiont-derived carbon and nitrogen among host sponges; therefore, these associations likely mediate the ecological diversification of coexisting sponge species. The goal of this project is to test this transformative hypothesis by adopting an integrative approach to assess the individual components of holobiont metabolism (i.e., microbial symbionts and sponge host) in ten of the most common sponge species in the Caribbean. The investigators will isolate autotrophic and heterotrophic metabolic pathways and explore potential links between microbial symbiont community composition and the assimilation of particulate and dissolved organic matter (POM and DOM) from seawater. This project will elucidate whether Caribbean sponge species are on similar or divergent evolutionary trajectories, and will provide information that is critical for our understanding of how conditions in the Caribbean basin have shaped the evolution of benthic organisms.</p>
Ecological Diversification in Sponges
largerWorkCitation
project
eng; USA
oceans
-81.40839451
-81.40839451
24.56381897
24.56381897
2021-08-01
2021-08-01
Caribbean coast of Panama
0
BCO-DMO catalogue of parameters from Bulk isotope data from sponges collected in Summerland Key in Florida between July 27 - August 19, 2021
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/954483.rdf
Name: Experiment
Units: unitless
Description: <p>Autotrophy dark, Autotrophy light, dissolved organic matter, or particulate organic matter refer to experiments measuring uptake of inorganic nutrients in dark, inorganic nutrients in light, dissolved organic matter, and particulate organic matter, respectively.</p>
http://lod.bco-dmo.org/id/dataset-parameter/954484.rdf
Name: time
Units: unitless
Description: <p>Timepoint at which samples collected (in hours) during experiment. T=0 samples were collected prior to experimentation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/954485.rdf
Name: replicate
Units: unitless
Description: <p>Replicate number; typically n=4 for each species at each timepoint in each experiment.</p>
http://lod.bco-dmo.org/id/dataset-parameter/954486.rdf
Name: species_abbreviation
Units: unitless
Description: <p>Abreviated codes for species names of each sample. Acau = Aplysina cauliformis, Ibir = Iotrochota birotulata, Ndig = Niphates digitalis, Xmut = Xestospongia muta.</p>
http://lod.bco-dmo.org/id/dataset-parameter/954487.rdf
Name: Species_name
Units: unitless
Description: <p>Scientific name</p>
http://lod.bco-dmo.org/id/dataset-parameter/954488.rdf
Name: Collection_date
Units: unitless
Description: <p>Collection date of sponge samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954489.rdf
Name: lat
Units: decimal degrees
Description: <p>Latitude of collection site for sponge individuals, west is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/954490.rdf
Name: lon
Units: decimal degrees
Description: <p>Longitude of collection site for sponge individuals, south is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/954491.rdf
Name: Linear_corr_d15N
Units: permille (‰)
Description: <p>Delta 15N value for each sample</p>
http://lod.bco-dmo.org/id/dataset-parameter/954492.rdf
Name: wt_perc_N
Units: percentag (%)
Description: <p>Weight percent of N in sample</p>
http://lod.bco-dmo.org/id/dataset-parameter/954493.rdf
Name: Atm_perc_15N
Units: percentag (%)
Description: <p>Atom percent 15N (of total N)</p>
http://lod.bco-dmo.org/id/dataset-parameter/954494.rdf
Name: Linear_corr_d13C
Units: permille (‰)
Description: <p>Delta 13C value for each sample</p>
http://lod.bco-dmo.org/id/dataset-parameter/954495.rdf
Name: wt_perc_C
Units: percentag (%)
Description: <p>Weight percent of C in sample</p>
http://lod.bco-dmo.org/id/dataset-parameter/954496.rdf
Name: Atm_perc_13C
Units: percentag (%)
Description: <p>Atom percent 13C (of total C)</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
56718
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/ac5676eb-ff23-4f72-a36e-b4eba70623f8/content
download
https://doi.org/10.26008/1912/bco-dmo.954470.1
download
onLine
dataset
<p>Sample collection<strong><em>:</em></strong>&nbsp;Fieldwork was conducted at Mote Marine Laboratory in Summerland Key,&nbsp;FL, USA, between July 27 and August 19, 2021. Replicate individuals of the four sponge species&nbsp;<em>Aplysina cauliformis</em>,&nbsp;<em>Iotrochota birotulata</em>,&nbsp;<em>Niphates digitalis</em>, and&nbsp;<em>Xestospongia muta</em>&nbsp;were collected from a shallow (7 m depth) patch reef (24°33’ N, 81°24’ W) offshore of Summerland Key (24°39’ N, 81°26’ W) via dive knives (with the spongocoel intact for the barrel/tube sponges by cutting the sponge from the substrate) using SCUBA, and were placed into plastic bags filled with seawater before being brought to the surface. After collection, all sponges were kept submerged in ambient seawater in large, sealed plastic bags in an insulated cooler during transit back to the lab. Upon return to Mote Marine Laboratory, sponges were attached with cable ties to plastic window screens, and held in large, flow-through raceways for 24 h for acclimatization. At no point during this process were sponges exposed to air.&nbsp;</p>
<p>Only healthy sponges (sponges confirmed to be actively pumping via use of fluorescein dye, and without any signs of necrosis) were processed for and used in experiments. For each experiment (and each treatment in the autotrophy experiment), four large individuals of each of the rope sponges&nbsp;<em>I. birotulata</em>&nbsp;and&nbsp;<em>A. cauliformis</em>&nbsp;collected from the reef were partitioned into seven subsamples for sampling at discrete time points. Both&nbsp;<em>X. muta</em>&nbsp;and&nbsp;<em>N. digitalis</em>&nbsp;were only sampled at four timepoints during each experiment because they have barrel/tube morphologies that cannot be successfully partitioned and subsampled, requiring the sacrifice of an entire individual for each timepoint. Therefore, sixteen small individuals of each of these two species were collected from the reef for each experiment. Experimental replicates were defined as follows: a replicate of&nbsp;<em>X. muta</em>&nbsp;consisted of one individual attached to a plastic window screen, which was placed in an 8 L, clear food-grade container; a replicate of&nbsp;<em>N. digitalis</em>&nbsp;consisted of four small individuals attached to a plastic window screen, which was placed into a 17.9 L, clear food-grade container; and replicates of&nbsp;<em>A. cauliformis</em>&nbsp;and&nbsp;<em>I. birotulata</em>&nbsp;consisted of all seven subsamples from the same individual attached to a plastic window screen, which was placed into a 17.9 L, clear food-grade container. In total, there were four replicates of each species collected at each time point in each experiment, with the exception the DOM experiment, during which multiple individuals, including all individuals of&nbsp;<em>A. cauliformis</em>,<em>&nbsp;</em>developed necroses in experimental tanks after 24h. The effected individuals were eliminated, limiting replication to only two timepoints (24h and 72h) during the chase for all species. Due to necroses of all individuals of&nbsp;<em>A. cauliformis&nbsp;</em>after 24h, the DOM experiment was repeated for this species only, using three healthy replicates.</p>
<p>Pulse-chase experiments<strong><em>:</em></strong>&nbsp;Three separate “pulse-chase” experiments were carried out to track the uptake and recycling of labeled compounds from three different resource pools. The first resource pool, DOM, consisted of&nbsp;<sup>13</sup>C-labeled glucose (35 μM) and galactose (35 μM), and&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N-labeled cell-free amino acids (range from 39-790 nM) and urea (2.2 μM). The second resource pool, POM, consisted of&nbsp;<sup>15</sup>N- and&nbsp;<sup>13</sup>C-labeled&nbsp;<em>Synechococcus&nbsp;</em>spp. bacteria incubated for 36 h in seawater containing 1.18 μM NaH<sup>13</sup>CO₃, 3.67 μM&nbsp;<sup>15</sup>NH₄Cl, and 0.117 M Na<sup>15</sup>NO₃, and&nbsp;<em>Oceanicola batsensis&nbsp;</em>incubated in 0.55 μM galactose, 1.18 μM NaH<sup>13</sup>CO₃, 3.67 μM&nbsp;<sup>15</sup>NH₄Cl, and 0.117 μM Na<sup>15</sup>NO₃. Finally, the third resource pool of inorganic compounds consisted of Na<sup>15</sup>NO₃ (0.117 μM) and NaH<sup>13</sup>CO₃ (1.18 μM). Labeled bacterial cells were added to filtered seawater for the POM experiment at a final combined concentration of 4.65 x10<sup>5</sup>&nbsp;cells ml<sup>-1</sup>&nbsp;for photosynthetic cells and 1.01 x 10<sup>6</sup>&nbsp;for heterotrophic cells (measured at the Center for Aquatic Cytometry at Bigelow Laboratory for Ocean Sciences). Concentrations of labeled resources were chosen to approximate the concentrations of corresponding inorganic and organic sources of carbon and nitrogen found in Caribbean seawater as closely as possible, as well as to match concentrations used in previous studies. In all incubations, experimental water was 0.7 μm (GF/F) filtered prior to the addition of tracers or labeled bacteria. Sponges were “pulsed” for 3 h in water containing these isotopically enriched resources, and then sponges were held in unenriched, flowing seawater for a 72-h “chase” period. Experimental samples were collected during the pulse at t=0.5 h, and during the chase at final times of t=12 h, 48 h, and 75 h (minus the 48h sampling in the DOM experiment). Prior to the start of the pulse, one of the seven subsamples of&nbsp;<em>A. cauliformis</em>&nbsp;and&nbsp;<em>I. birotulata</em>, and four individuals each of&nbsp;<em>X. muta</em>&nbsp;and&nbsp;<em>N. digitalis</em>&nbsp;were sampled and processed to serve as t = 0 h (natural abundance) samples.</p>
<p>To start the pulse, each experimental replicate was added to a food grade container containing filtered seawater and labeled compounds or food bacteria, as well as a recirculating pump used to maintain flow. Replicates of&nbsp;<em>X. muta</em>were held in 8 L, clear food grade containers holding 6 L of filtered seawater, while replicates of&nbsp;<em>I. birotulata</em>,&nbsp;<em>A. cauliformis</em>, and&nbsp;<em>N. digitalis</em>&nbsp;were held in 17.9 L containers holding 15 L of filtered seawater. These containers were all held in a large 4.6 m-diameter outdoor tank with a shallow seawater bath to control temperature in experimental tanks holding sponges. At the end of the pulse, the water level in the outdoor tank was raised to flood experimental tanks with unlabeled seawater (supplied from the canal adjacent to Mote Marine Lab, drawn from 10 m, and filtered through sand to 40 μm) throughout the chase. Temperature measurements were taken at regular intervals (30 min), and an additional 25°C supply of well water, with salinity equal to that of seawater, was supplemented to maintain a tank temperature between 29-32°C. During the day for all experiments, irradiance measurements were taken every 30 min to monitor for deviation outside of the irradiance that sponges experience at depth, which was 800 to 1000 μmol photons m<sup>-2</sup>&nbsp;s<sup>-1</sup>&nbsp;measured at depth during cloudless daytime conditions at the collection site. One layer of standard shade cloth was sufficient to keep irradiance within the normal limits of what the sponges experience at depth, and adjustments during the experiment were only made when irradiance went below the 800-1000 μmol photons m<sup>-2</sup>&nbsp;s<sup>-1</sup>&nbsp;range under cloudy conditions. When irradiance decreased, we removed the layer of shade cloth to increase exposure of the sponges to ambient sunlight.</p>
<p>To assess the role of both autotrophic and heterotrophic symbionts in the assimilation of inorganic compounds, the autotrophy experiment included both light and dark treatments. For this reason, each large individual of&nbsp;<em>I. birotulata</em>&nbsp;and&nbsp;<em>A. cauliformis&nbsp;</em>was partitioned into 14 subsamples, and 32 small individuals each of&nbsp;<em>N. digitalis&nbsp;</em>and&nbsp;<em>X. muta</em>&nbsp;were collected for this experiment. Half of the subsamples or individuals of each species were randomly assigned to a dark treatment (covered in opaque plastic tarp with irradiance values from HOBO loggers that detected 0 Lux throughout the pulse), and the other half to a light treatment (exposure to ambient irradiance as in other experiments). Samples in the dark treatment were covered during the pulse but were exposed to ambient irradiance during the chase so that we could monitor nutrient retention and recycling under ambient (light-dark cycle) conditions.</p>
<p>Sample fixation<strong><em>:</em></strong>&nbsp;After sampling during pulse-chase experiments, sponges were rinsed with 0.7 μm-filtered, unenriched seawater and lightly blotted with a paper towel before weighing to the nearest 0.001 g to obtain a wet weight. Afterwards, ~2 mm cross sections were collected in duplicate using a razor blade and placed into 2 mL cryovials to be fixed for SEM and NanoSIMS analysis; the remaining bulk tissue was frozen at -20°C for isotope analysis. The 2 mm cross-sections of sponge tissue were fixed in&nbsp;2.5% (w/v) glutaraldehyde + 1% (w/v) paraformaldehyde in 1.5X&nbsp;PHEM buffer (1.5X PHEM (90 mM PIPES, 37.5 mM HEPES, 15 mM EGTA, 3 mM&nbsp;MgSO4.7H<sub>2</sub>O), and 9% (w/v) sucrose at pH 7.4) and stored at 4°C&nbsp;for 12h before being rinsed 3x with PHEM buffer containing no fixative. Finally, 0.1 mL of fixative solution (10% of the concentration in the original solution) was added back to each sample to prevent any fouling during long-term storage at&nbsp;4°C.&nbsp;After being taken out of storage,&nbsp;cross-sections of sponge tissue were rinsed once more in fixative-free 1.5X PHEM buffer, digested in 5% hydrofluoric acid to break down siliceous spicules within the sponge tissue (note this was carried out for all samples to be consistent, even though&nbsp;<em>A. cauliformis</em>&nbsp;lacks spicules), and rinsed twice and stored in 1.5X PHEM buffer.</p>
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<p>Bulk stable isotope analysis: To prepare for bulk isotope analysis, a small subsample of frozen tissue from each replicate of each species was placed into a scintillation vial and lyophilized overnight to remove seawater. Dried samples were homogenized with a mortar and pestle, and homogenized tissue was acidified with 12N HCl fumes in an acid chamber to remove carbonate before being weighed into tin capsules to the nearest 0.0001 mg using a Mettler Toledo XPR microbalance at Hollings Marine Lab in Charleston, SC, USA. Samples were analyzed at the Smithsonian MCI Stable Isotope Mass Spectrometry Laboratory using a Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar Vario ISOTOPE Cube Elemental Analyzer via a Thermo Conflo IV. Enrichment is expressed in delta notation (δ<sup>13</sup>C and δ<sup>15</sup>N) in units of permille.</p>
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* split latitude & longitude into own column
* converted date to iso format (yyyy-mm-dd)
* Adjusted parameter names to comply with database requirements
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