http://lod.bco-dmo.org/id/dataset/954735
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
2025-02-27
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Autotrophy, heterotrophy and niche partitioning in Caribbean sponges sampled June 9, 2019 on reef sites around Bocas del Toro Panama.
2025-02-27
publication
2025-02-27
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2025-05-07
publication
https://doi.org/10.26008/1912/bco-dmo.954735.1
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., Parry, A., Houk, J. (2025) Autotrophy, heterotrophy and niche partitioning in Caribbean sponges sampled June 9, 2019 on reef sites around Bocas del Toro Panama. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2025-02-27 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.954735.1 [access date]
Autotrophy, heterotrophy, and niche partitioning Dataset Description: Methods and Sampling: <p>In June of 2019, individuals of the sponge species&nbsp;<em>Aiolochroia crassa</em>,&nbsp;<em>Amphimedon compressa, Aplysina cauliformis, Aplysina fulva, Iotrochota birotulata, Ircinia bocatorensis&nbsp;</em>(formerly&nbsp;<em>Ircinia felix</em>; Kelly &amp; Thacker 2021)<em>, Mycale laevis, Niphates erecta, Verongula rigida,&nbsp;</em>and<em>&nbsp;Xestospongia muta</em>&nbsp;were collected by SCUBA from shallow reefs (3-10 m) around the Smithsonian Tropical Research Institute (STRI) in the Bocas del Toro (BDT) archipelago of Panama.&nbsp;&nbsp;Individuals of the species&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida&nbsp;</em>in BDT can be easily partitioned into genetically identical fragments that can be allocated to multiple experiments or treatments. In contrast, the species&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;cannot be reliably split into multiple individuals. Thus, to start these experiments, seven large individuals of&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida</em>&nbsp;were collected. Each individual of these species was partitioned into four fragments (an initial sample and one sample each for the heterotrophy experiment and light and dark treatments of the autotrophy experiment). Small individuals (N = 28 each) of&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;were also collected and these whole individuals were used in each experiment. All fragments and intact individuals were labeled and initially attached to a small square of plastic window screening that was cable tied to plastic mesh secured to the benthos with cinder blocks. Sponges were acclimated in this shallow (2-3 m) common garden reef near STRI for at least 48 hr before the start of experiments. After 48 hr and prior to the start of experiments, initial samples of each species (N = 7) were removed from this common garden and processed for microbiome (small subsample placed in 95 % EtOH and frozen at -20 °C) and isotope (wrapped in aluminum foil and frozen at -20 °C) analyses.</p>
<p>Prior to the start of the experiment, irradiance was measured at multiple depths from collection sites around STRI using a LI-COR LI-193 underwater spherical quantum sensor. Vertical light extinction coefficients averaged 0.355, indicating that our average irradiance (between 10 am and 4 pm) of over 3000&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>&nbsp;at the surface (as measured by the&nbsp;STRI weather platform; Paton 2019) corresponded to over 500&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>&nbsp;at 5 m depth.</p>
<p>The experiment was carried out for seven days in a Latin square design with one replicate of each of the ten sponge species used per day. During the evening prior to the experiment, sponges were collected from the common garden reef and dark-adapted overnight in a common flow-through seawater tank covered in thick plywood. At the start of the experiment, sponges were transferred to either 4 L or 6 L (for larger&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;individuals) experimental containers filled with 0.7 µm (GF/F) filtered seawater that was dosed with NaH<sup>13</sup>CO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>) and Na<sup>15</sup>NO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>) as in Freeman et al. (2013). These containers were resting within flowing seawater tables that acted as a temperature bath (Fig. S1). Two fragments of a replicate of&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida&nbsp;</em>or individuals of&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;were each relegated to an experimental container in one of two flowing seawater tables: a light treatment that was exposed to direct, ambient light and a dark treatment that was under the roof of the wet lab at STRI and covered with a sheet of thick plywood. Light loggers placed within this dark tank during all seven days of the experiment consistently measured zero irradiance. Water circulation in each experimental container was maintained using a submersible pump. Water temperature in experimental containers was measured every 30 min and, if needed, ice packs were added to the temperature bath to maintain a target temperature of 29-30 °C. Ambient irradiance was monitored every 15 min using the STRI weather station (Paton 2019; in irradiance units of&nbsp;W m<sup>-2</sup>), and layers of window screening were added to the light treatment as needed in order to maintain an irradiance level in the experimental containers that approached irradiance at 3 m depth (1000&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>) where almost all of our species were found. A conversion factor of 1 W m<sup>-2</sup>&nbsp;to 457&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1&nbsp;</sup>was used for this calculation. At the end of the experiment (6 hr), sponge tissue was rinsed in 0.7 µm filtered, unlabeled seawater, a small subsample was removed for microbiome analyses and preserved in 95% EtOH as outlined above, and the remaining tissue was wrapped in aluminum foil, and frozen at -20 °C for transport back to Charleston, SC. Sponges were later weighed to the nearest 0.001 g after thawing.&nbsp;</p>
<p>The bacterium&nbsp;<em>Oceanicola batsensis</em>&nbsp;(NCMA B15&nbsp;from&nbsp;https://ncma.bigelow.org/) was purchased to serve as the primary bacterium for the heterotrophy experiment, but local heterotrophic bacteria were also cultured from the water near STRI. To isolate ambient bacteria, 250 mL of seawater from the site’s flow through system (that acquires water from a seagrass and hardbottom habitat approximately 100 m offshore) was filtered through a 0.22 µm polycarbonate filter. Sterile techniques were employed in a countertop, portable hood sterilized with 70% EtOH. The filter was placed into sterile 15 ml conical tubes with 8 ml of 0.2 µm filtered seawater and shaken gently. The filter was plated face down, and the water was streaked out onto the following agar preparations: 1.5% Difco marine broth 2216 with 0.2 µm unamended filtered sea water (FSW) and 1.5 % Difco marine broth 2216 with 0.7 µm FSW 1.5 % + 10 mM NH<sub>4</sub>Cl. These culturing techniques led to the formation of two colonies: JHB59-1.1 was a yellow, smooth colony isolated on 1.5% Difco marine broth 2216, and JHB59-3.2 were clear colonies isolated on 0.2 µm filtered seawater 1.5 % + 10 mM NH<sub>4</sub>Cl. Each of these, along with the purchased&nbsp;<em>O. batsensis</em>, were enriched in&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N for the heterotrophy experiments to measure sponge cell isotopic enrichment as a proxy for cell removal and assimilation.&nbsp;</p>
<p>&nbsp;A day prior to each experiment, colonies of&nbsp;<em>O. batsensis</em>, JHB59-1.1, and JHB59-3.2 were used to create starter cultures with 4 ml of marine broth. The evening prior to the experiment, the starter culture was added to 400 ml of marine broth, and the media was amended with&nbsp;<sup>13</sup>C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>&nbsp;(Glucose) (0.4. g L<sup>-1</sup>), NaH<sup>13</sup>CO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>),&nbsp;<sup>15</sup>NH<sub>4</sub>Cl (0.2 g L<sup>-1</sup>), and Na<sup>15</sup>NO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>). Dual carbon and nitrogen labels were used because one strain of&nbsp;<em>O. batsensis</em>&nbsp;does not utilize glucose and to ensure adequate&nbsp;<sup>15</sup>N enrichment. All isotopic tracer compounds had an atom % of the heavy atom of C and N (<sup>13</sup>C and&nbsp;<sup>15</sup>N) of at least 98%. Cultures were grown in an incubator at 29°C with rocking motion. On the morning of the experiment, the OD600 concentration was recorded for the overnight cultures using a spectrophotometer and then cells were pelleted by centrifugation in 50 ml Falcon tubes at 4500 x g for 10 min. Cells were resuspended in 12 ml of 0.7 µm filtered seawater for use in the experiment. Each day, the remainder of culture was stored in RNAlater and stored at -20 °C for future microbial 16S rDNA analysis.</p>
<p>The heterotrophy experiment was conducted over four consecutive days in a Latin square design with at least one replicate (one replicate on day #1 and two replicates in days two, three, and four) of each of the ten sponge species used per day. On the evening prior to the experiment, sponges were collected from the common garden reef and dark-adapted overnight in a common flow-through seawater tank. As above, at the start of the experiment, sponges were transferred to either 4 L or 6 L experimental containers filled with 0.7 µm (GFF) filtered seawater resting within a flow table with running seawater, and water circulation in experimental containers was maintained using a submersible pump for 120 min. Sponges were dosed with&nbsp;<sup>13</sup>C- and&nbsp;<sup>15</sup>N-enriched bacteria using a pipette at an initial average concentration of 3x10<sup>6&nbsp;</sup>microbial cells per ml. An initial 1 ml water sample was collected immediately at T = 0 for future flow cytometry analysis and this was repeated at 30 min intervals for T = 30, 60, 90, and 120 min. Data from the 90 min sampling were ultimately excluded from the study due to preservation issues. Samples were also collected from a control (without a sponge) tank dosed with the same concentration of bacteria as the sponge tanks each day; one control tank was included on day 1 and two controls tanks were included in days 2, 3, and 4 of the experiment. All water samples were added to a 2 ml screw top cryovial tube with 0.5% paraformaldehyde (final concentration), held at 4 °C for one hr, and then frozen at -20 °C. Water temperature was recorded every 30 min and ice packs were added to the surrounding water bath as needed. After 120 min of incubation with the enriched bacteria, flowing seawater was added to each tank via individual plastic tubing and tanks were flushed with ambient water for one hr to allow for the purging of enriched bacteria that might be transiting the sponge aquiferous system but had not yet been consumed. Following this, sponge tissue was rinsed in 0.7 µm filtered, unlabeled seawater, a small subsample was removed for microbiome analyses and preserved in 95% EtOH as outlined above, and the remaining tissue was wrapped in aluminum foil, and frozen at -20 °C for transport back to Charleston, SC. Sponges were later weighed to the nearest 0.001 g after thawing.&nbsp;</p>
<p>To determine the clearance rate for all ten sponge species, heterotrophic bacteria abundance was enumerated at each time point using flow cytometry. Cell counting was performed using a BD Biosciences Accuri C6 Plus Model flow cytometer at the Smithsonian Marine Station in Fort Pierce, Florida. The methods for staining heterotrophic bacteria with SYBR Green 1. Each day, fluoresce bright Plain YG 1.0 µm beads were added at 1.0 x 10<sup>6</sup>&nbsp;to 0.2 µm filtered seawater and run as a size standard, and a blank consisting of 1 ml TE (10 mM Tris-HCL, 1 mM EDTA) buffer and 10 µl 50x SYBR Green was also analyzed to differentiate signal from the TE buffer (distinct crescent shape output on the display) from the signal of experimental samples.</p>
<p>Each 1 ml experimental water sample was thawed and vortexed to ensure bacterial cells were fully resuspended. 100 µl of each sample was added to 900 µl of TE buffer and 10 µl of 50x SYBR Green in a new 1.5 ml centrifuge tube. This tube was inverted, incubated in a -20 °C freezer for 15 min in the dark, and run on the BD C6 flow cytometer at a flow rate of 35 μl min<sup>-1</sup>&nbsp;for 2 min. Samples were excited by a 488 nm laser and cell populations were detected using a 533/30 filter (FL-1), forward and side scatter with a threshold of 700 on FL-1. To reduce potential contamination across samples, the flow cytometer was backflushed, rinsed for one min with milliQ water at high flow rate (66 μl min<sup>-1</sup>), and the sample nozzle was wiped in between each sample. If a water sample (experimental or control) was analyzed more than once, then an average concentration of cells from all runs was ultimately used for a concentration value in the equations below.&nbsp;</p>
<p>The isolation of microbial and sponge cells from bulk sponge tissue followed methods adapted from Freeman et al. (2013). Sponge tissue was minced into small pieces using a sterile razor blade, added to 50 ml falcon tubes, and the sponge pieces were allowed to thaw for an hr at room temperature before being covered with ice-cold calcium-magnesium-free artificial seawater (CMFASW) amended with&nbsp;Ethylenediaminetetraacetic acid&nbsp;(EDTA; as in Freeman &amp; Thacker 2011). Samples were gently homogenized with a mortar and pestle 55x to dissociate the sponge and bacterial cells from the sponge matrix. This solution was filtered through 50 µm Nitex nylon mesh and lightly squeezed to retain undissociated cells and large spicules.</p>
<p>The resulting filtrate was poured into 50 ml centrifuge tubes and topped with CMFASW-EDTA for a final concentration of 50 ml. The filtrate for each sponge species was centrifuged at low speed to first isolate sponge cells and then high speeds to isolate microbial cells. To optimize sample purity, unique methods and speeds were used for each species (as in Freeman &amp; Thacker 2011, Freeman et al. 2013; Table S3). To isolate initial sponge cell fractions (containing some microbial cells), the above filtrate was centrifuged, and the supernatant after two to four low-speed rounds of centrifugation was decanted into a new 50 ml Falcon tube that was then centrifuged at 8000 RCF to obtain a microbial pellet (Table S3). Following this high-speed spin, the supernatant was poured off and the microbial pellet was stored at -20 °C. The initial pellets from low-speed centrifugation contained primarily sponge cells but needed to be purified again at lower speeds to remove microbial cells. Sponge pellets from consecutive low-speed centrifugation were resuspended in 15 ml of CMFASW-EDTA and combined. After each sponge pellet cleaning spin, the supernatant was discarded, and the cells were resuspended in 15 ml CMFASW-EDTA for the following spins. Purified sponge and microbial cell pellets were transferred to 2 ml micro-centrifuge tubes and centrifuged at 7200 RCF for 5 min at 4 °C. The supernatant was discarded, and the pellets were stored at -20 °C. Bulk sponge tissue was used for&nbsp;<em>Amphimedon compressa&nbsp;</em>because a pure sponge pellet could not be obtained due to cells rupturing during the dissociation process and because&nbsp;<em>A. compressa&nbsp;</em>has a very low abundance of microbial cells such that the microbial cell pellets lacked the biomass required for isotope analyses. Sponge and microbial fraction purity were verified using fluorescence and phase contrast microscopy at 400X (as in Freeman &amp; Thacker 2011).&nbsp;</p>
<p>The purified and frozen sponge and microbial fractions and subsamples of&nbsp;<em>A. compressa&nbsp;</em>tissue were lyophilized for 24 hr. Dried&nbsp;<em>A. compressa&nbsp;</em>was crushed with a mortar and pestle into a fine powder and transferred into 1.5 ml centrifuge tubes, and all dried pellets were placed in a 65 °C drying oven for 12 hr to remove any residual moisture. Samples were then acidified to remove any carbonate by exposure to 12 mol L<sup>-1</sup>&nbsp;HCl vapors for 12 hr in a sealed desiccator. Residual acid was removed following acidification by placing samples back into a 65 °C drying oven for 12 hr. Dried samples were weighed into tared tin capsules (Costech) to the nearest 0.001 mg (1.8 – 2.0 mg) using a microbalance.&nbsp;Crushed capsules were arranged into a 96-well plate and submitted for stable isotope analysis&nbsp;at the Smithsonian Institution OUSS/MCI Stable Isotope Mass Spectrometry Facility in&nbsp;Suitland, MD.&nbsp;Isotope ratios (δ<sup>13</sup>C and δ<sup>15</sup>N) of each fraction were measured on a Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV. All calculations of raw isotope values are performed with Isodat 3.0 software. Analytical precision at the Smithsonian Institution OUSS/MCI Stable Isotope Mass Spectrometry Facility is ± 0.2 ‰.&nbsp;</p>
<p>Sponge and microbial cell isotopic (<sup>15</sup>N and&nbsp;<sup>13</sup>C) enrichment was initially expressed in delta (δ) notation in permil units (<sup>0</sup>‰; in&nbsp;δ<sup>13</sup>C and δ<sup>15</sup>N values) but because initial natural abundance values were available for all species, enrichment for both experiments was ultimately expressed as change (Δ) in δ<sup>13</sup>C and δ<sup>15</sup>N (Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N value when compared to initial samples). These initial and experimental samples were matched for sponge genotype (same individual split among initial and experimental samples for&nbsp;<em>A. cauliformis</em>,&nbsp;<em>A. crassa</em>,&nbsp;<em>A. fulva</em>,&nbsp;<em>I. birotulata</em>,&nbsp;<em>M. laevis, N. erecta</em>,&nbsp;<em>V. rigida</em>, but not for&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;because these sponges cannot be easily and successfully partitioned into multiple individuals. Thus, for&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>, all seven initial (non-experimental) replicates were pooled to obtain an average natural abundance value for sponge and microbial cell fractions of each species. In addition, due to sample loss, there were only two initial&nbsp;<em>A. compressa</em>&nbsp;tissue samples; these two samples were used to obtain an initial average δ<sup>13</sup>C and δ<sup>15</sup>N value of holobiont tissue. These average initial δ<sup>13</sup>C and δ<sup>15</sup>N values were then subtracted from the δ<sup>13</sup>C and δ<sup>15</sup>N values of microbial and sponge cell fractions (or holobiont for&nbsp;<em>A. compressa</em>) from individual experimental replicates to obtain&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N values for&nbsp;<em>A. compressa</em>,&nbsp;<em>I. bocatorensis</em>, and&nbsp;<em>X.</em>&nbsp;<em>muta</em>.</p>
<p>Because&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the light treatment was derived from both autotrophic and heterotrophic microbial metabolism, but&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the dark treatment was only influenced by heterotrophic microbes, we isolated the role of autotrophic microbes in holobiont C and N metabolism in the light by subtracting&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment (calculated as&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N) in the dark treatment from total&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment (Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N) in the light treatment for each individual sponge sample. These values were ultimately expressed as % phototrophic and % heterotrophic metabolism according to the contribution of each pathway to total&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the light. Because&nbsp;<em>I. bocatorensis&nbsp;</em>and&nbsp;<em>X. muta</em>&nbsp;samples were not matched (the same genotype was not used in both light and dark experiments),&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment by phototrophic symbionts was measured by subtracting the average&nbsp;Δδ<sup>13</sup>C andΔδ<sup>15</sup>N values in the dark from average&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N values in the light.&nbsp;</p>
<p>Heterotrophic efficiency of particulate organic matter cell removal was calculated using clearance rate at an intermediate (60 min) and final (120 min) time point. Clearance rate (CR) represents the volume of water cleared of suspended particles per unit of time and is corrected by mass of the sponge according to the equation:</p>
<p>CR= (V/t<sub>x</sub>m) ln(C<em><sub>o</sub></em>/C<em><sub>t</sub></em>)</p>
<p>where V<sub>&nbsp;&nbsp;</sub>is the volume of the experimental tanks in ml (4000 ml or 6000 ml), t is time in hr (one or two hr), m is the wet mass of the sponge, C<sub>0</sub>&nbsp;is the starting concentration of bacteria in the tank, and C<sub>t</sub>&nbsp;is the concentration of bacteria at each time point (T = 60 min and T = 120 min). Because the concentration of labeled heterotrophic bacteria in control tanks varied minimally over the 120 min incubations with no significant decrease in cell number, clearance rates in experimental tanks were not corrected based on controls&nbsp;</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
Species
Full_scientific_name
Date
Lat
Lon
Replicate
Fraction
Initial_d15N
Initial_wt_percN
Initial_Atm_perc15N
Initial_d13C
Initial_wt_percC
Atm_perc13C
Dark_d15N
Dark_wt_percN
Dark_Atm_perc15N
Dark_d13C
Dark_wt_percC
Dark_Atm_perc13C
Dark_Change_in_d15N
Dark_Change_in_d13C
Light_d15N
Light_wt_percN
Light_Atm_perc15N
Light_d13C
Light_wt_percC
Light_Atm_perc13C
Light_Change_in_d15N
Light_Change_in_d13C
Light_photo_d15N
light_photo_d13C
Hetero_d15N
hetero_d13C
POM_heterotrophy_enriched_d13C
POM_heterotrophy_enriched_d15N
Heterotrophy_POM_change_in_d13c
Heterotrophy_POM_change_in_d15N
POM_T60_CR
POM_T120_CR
BD Biosciences Accuri C6 Plus Model
Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV
LI-COR LI-193 underwater spherical quantum sensor
theme
None, User defined
species scientific name (binomial)
date
latitude
longitude
replicate
sample fraction (includes size fractions)
d15N
Nitrogen
15N, stable isotope of N
d13C
Elemental carbon (C)
13C, stable isotope of C
flow rate
featureType
BCO-DMO Standard Parameters
Flow Cytometer
Mass Spectrometer
Photosynthetically Available Radiation Sensor
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: 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
-82.262448
-82.262448
9.349457
9.349457
2019-06-09
2019-06-09
Caribbean coast of Panama
0
BCO-DMO catalogue of parameters from Autotrophy, heterotrophy and niche partitioning in Caribbean sponges sampled June 9, 2019 on reef sites around Bocas del Toro Panama.
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/954766.rdf
Name: Species
Units: unitless
Description: <p>Sponge species (Acau: Aplysina cauliformis; Acra: Aiolochroia crassa; Aful: Aplysina fulva; Acom: Amphimedon compressa; Ibiro: Iotrochota birotulata; Ifel: Ircinia felix; Mlae: Mycale laevis; Nere: Niphates erecta; Vrig: Verongula rigida; Xmut: Xestospongia muta).</p>
http://lod.bco-dmo.org/id/dataset-parameter/954767.rdf
Name: Full_scientific_name
Units: unitless
Description: <p>Complete scientific name of each sponge species</p>
http://lod.bco-dmo.org/id/dataset-parameter/954768.rdf
Name: Date
Units: unitless
Description: <p>Date of sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/954769.rdf
Name: Lat
Units: decimal degrees
Description: <p>Latitude of collection site, south is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/954770.rdf
Name: Lon
Units: decimal degrees
Description: <p>Longitude of collection site, west is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/954771.rdf
Name: Replicate
Units: unitless
Description: <p>Replicate number (1-10)</p>
http://lod.bco-dmo.org/id/dataset-parameter/954772.rdf
Name: Fraction
Units: unitless
Description: <p>Microbial cell fraction (Bac) or sponge cell fraction (Sponge) isolated from bulk sponge tissue; holobiont (combined bulk tissue including both microbial and sponge cells for Amphimedon compressa)</p>
http://lod.bco-dmo.org/id/dataset-parameter/954773.rdf
Name: Initial_d15N
Units: permille ( ‰)
Description: <p>Nitrogen isotope ratio in Initial, wild-caught samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954774.rdf
Name: Initial_wt_percN
Units: percentage (%)
Description: <p>Percent nitrogen content in initial, wild-caught samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954775.rdf
Name: Initial_Atm_perc15N
Units: percentage (%)
Description: <p>Percent 15N in initial, wild-cauight samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954776.rdf
Name: Initial_d13C
Units: permille ( ‰)
Description: <p>Carbon isotope ratio in Initial, wild-caught samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954777.rdf
Name: Initial_wt_percC
Units: percentage (%)
Description: <p>Percent carbon content in initial, wild-caught samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954778.rdf
Name: Atm_perc13C
Units: percentage (%)
Description: <p>Percent 13C in initial, wild-caught samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954779.rdf
Name: Dark_d15N
Units: permille ( ‰)
Description: <p>Nitrogen isotope ratio in samples from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954780.rdf
Name: Dark_wt_percN
Units: percentage (%)
Description: <p>Percent nitrogen content in samples from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954781.rdf
Name: Dark_Atm_perc15N
Units: percentage (%)
Description: <p>Percent 15N in samples from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954782.rdf
Name: Dark_d13C
Units: permille ( ‰)
Description: <p>Carbon isotope ratio in sample from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954783.rdf
Name: Dark_wt_percC
Units: percentage (%)
Description: <p>Percent carbon content in samples from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954784.rdf
Name: Dark_Atm_perc13C
Units: percentage (%)
Description: <p>Percent 13C in samples from dark experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954785.rdf
Name: Dark_Change_in_d15N
Units: permille ( ‰)
Description: <p>Change in nitgrogen isotope value in dark treatment relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954786.rdf
Name: Dark_Change_in_d13C
Units: permille ( ‰)
Description: <p>Change in carbon isotope value in dark treatment relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954787.rdf
Name: Light_d15N
Units: permille ( ‰)
Description: <p>Nitrogen isotope ratio in samples from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954788.rdf
Name: Light_wt_percN
Units: percentage (%)
Description: <p>Percent nitrogen content in samples from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954789.rdf
Name: Light_Atm_perc15N
Units: percentage (%)
Description: <p>Percent 15N in samples from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954790.rdf
Name: Light_d13C
Units: permille ( ‰)
Description: <p>Carbon isotope ratio in sample from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954791.rdf
Name: Light_wt_percC
Units: percentage (%)
Description: <p>Percent carbon content in samples from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954792.rdf
Name: Light_Atm_perc13C
Units: percentage (%)
Description: <p>Percent 13C in samples from light experimental treatment</p>
http://lod.bco-dmo.org/id/dataset-parameter/954793.rdf
Name: Light_Change_in_d15N
Units: permille ( ‰)
Description: <p>Change in nitgrogen isotope value in light treatment relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954794.rdf
Name: Light_Change_in_d13C
Units: permille ( ‰)
Description: <p>Change in carbon isotope value in light treatment relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954795.rdf
Name: Light_photo_d15N
Units: permille ( ‰)
Description: <p>Enrichment in 15N in the light treatment attributed to photosymbionts (total enrichment in the light-enrichment in the dark due to heterotrophic bacteria)</p>
http://lod.bco-dmo.org/id/dataset-parameter/954796.rdf
Name: light_photo_d13C
Units: permille ( ‰)
Description: <p>Enrichment in 13C in the light treatment attributed to photosymbionts (total enrichment in the light-enrichment in the dark due to heterotrophic bacteria)</p>
http://lod.bco-dmo.org/id/dataset-parameter/954797.rdf
Name: Hetero_d15N
Units: permille ( ‰)
Description: <p>Enrichment in 15N in the dark attributed to heterotrophic bacterial metabolism</p>
http://lod.bco-dmo.org/id/dataset-parameter/954798.rdf
Name: hetero_d13C
Units: permille ( ‰)
Description: <p>Enrichment in 13C in the dark attributed to heterotrophic bacterial metabolism</p>
http://lod.bco-dmo.org/id/dataset-parameter/954799.rdf
Name: POM_heterotrophy_enriched_d13C
Units: permille ( ‰)
Description: <p>Enrichment in 13C from consuming bacteria labeled in 13C</p>
http://lod.bco-dmo.org/id/dataset-parameter/954800.rdf
Name: POM_heterotrophy_enriched_d15N
Units: permille ( ‰)
Description: <p>Enrichment in 15N from consuming bacteria labeled in 15N</p>
http://lod.bco-dmo.org/id/dataset-parameter/954801.rdf
Name: Heterotrophy_POM_change_in_d13c
Units: permille ( ‰)
Description: <p>Change in carbon isotope value following consumption of 13C labeled bacterial cells relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954802.rdf
Name: Heterotrophy_POM_change_in_d15N
Units: permille ( ‰)
Description: <p>Change in nitgrogen isotope value following consumption of 15N labeled bacterial cells relative to initial samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/954803.rdf
Name: POM_T60_CR
Units: mililiters/hour
Description: <p>Clearance rate at 60 minutes in heterotrophy experiment based on cell counts via flow cytometry</p>
http://lod.bco-dmo.org/id/dataset-parameter/954804.rdf
Name: POM_T120_CR
Units: mililiters/hour
Description: <p>Clearance rate at 120 minutes in heterotrophy experiment based on cell counts via flow cytometry</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
55847
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/d19d6a37-0afa-4f49-b0cd-228f79b2705e/content
download
https://doi.org/10.26008/1912/bco-dmo.954735.1
download
onLine
dataset
<p>In June of 2019, individuals of the sponge species&nbsp;<em>Aiolochroia crassa</em>,&nbsp;<em>Amphimedon compressa, Aplysina cauliformis, Aplysina fulva, Iotrochota birotulata, Ircinia bocatorensis&nbsp;</em>(formerly&nbsp;<em>Ircinia felix</em>; Kelly &amp; Thacker 2021)<em>, Mycale laevis, Niphates erecta, Verongula rigida,&nbsp;</em>and<em>&nbsp;Xestospongia muta</em>&nbsp;were collected by SCUBA from shallow reefs (3-10 m) around the Smithsonian Tropical Research Institute (STRI) in the Bocas del Toro (BDT) archipelago of Panama.&nbsp;&nbsp;Individuals of the species&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida&nbsp;</em>in BDT can be easily partitioned into genetically identical fragments that can be allocated to multiple experiments or treatments. In contrast, the species&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;cannot be reliably split into multiple individuals. Thus, to start these experiments, seven large individuals of&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida</em>&nbsp;were collected. Each individual of these species was partitioned into four fragments (an initial sample and one sample each for the heterotrophy experiment and light and dark treatments of the autotrophy experiment). Small individuals (N = 28 each) of&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;were also collected and these whole individuals were used in each experiment. All fragments and intact individuals were labeled and initially attached to a small square of plastic window screening that was cable tied to plastic mesh secured to the benthos with cinder blocks. Sponges were acclimated in this shallow (2-3 m) common garden reef near STRI for at least 48 hr before the start of experiments. After 48 hr and prior to the start of experiments, initial samples of each species (N = 7) were removed from this common garden and processed for microbiome (small subsample placed in 95 % EtOH and frozen at -20 °C) and isotope (wrapped in aluminum foil and frozen at -20 °C) analyses.</p>
<p>Prior to the start of the experiment, irradiance was measured at multiple depths from collection sites around STRI using a LI-COR LI-193 underwater spherical quantum sensor. Vertical light extinction coefficients averaged 0.355, indicating that our average irradiance (between 10 am and 4 pm) of over 3000&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>&nbsp;at the surface (as measured by the&nbsp;STRI weather platform; Paton 2019) corresponded to over 500&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>&nbsp;at 5 m depth.</p>
<p>The experiment was carried out for seven days in a Latin square design with one replicate of each of the ten sponge species used per day. During the evening prior to the experiment, sponges were collected from the common garden reef and dark-adapted overnight in a common flow-through seawater tank covered in thick plywood. At the start of the experiment, sponges were transferred to either 4 L or 6 L (for larger&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;individuals) experimental containers filled with 0.7 µm (GF/F) filtered seawater that was dosed with NaH<sup>13</sup>CO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>) and Na<sup>15</sup>NO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>) as in Freeman et al. (2013). These containers were resting within flowing seawater tables that acted as a temperature bath (Fig. S1). Two fragments of a replicate of&nbsp;<em>A. crassa</em>,&nbsp;<em>A. compressa, A. cauliformis, A. fulva, I. birotulata, M. laevis, N. erecta,&nbsp;</em>and&nbsp;<em>V. rigida&nbsp;</em>or individuals of&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;were each relegated to an experimental container in one of two flowing seawater tables: a light treatment that was exposed to direct, ambient light and a dark treatment that was under the roof of the wet lab at STRI and covered with a sheet of thick plywood. Light loggers placed within this dark tank during all seven days of the experiment consistently measured zero irradiance. Water circulation in each experimental container was maintained using a submersible pump. Water temperature in experimental containers was measured every 30 min and, if needed, ice packs were added to the temperature bath to maintain a target temperature of 29-30 °C. Ambient irradiance was monitored every 15 min using the STRI weather station (Paton 2019; in irradiance units of&nbsp;W m<sup>-2</sup>), and layers of window screening were added to the light treatment as needed in order to maintain an irradiance level in the experimental containers that approached irradiance at 3 m depth (1000&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1</sup>) where almost all of our species were found. A conversion factor of 1 W m<sup>-2</sup>&nbsp;to 457&nbsp;µmol photons m-<sup>2</sup>&nbsp;s<sup>-1&nbsp;</sup>was used for this calculation. At the end of the experiment (6 hr), sponge tissue was rinsed in 0.7 µm filtered, unlabeled seawater, a small subsample was removed for microbiome analyses and preserved in 95% EtOH as outlined above, and the remaining tissue was wrapped in aluminum foil, and frozen at -20 °C for transport back to Charleston, SC. Sponges were later weighed to the nearest 0.001 g after thawing.&nbsp;</p>
<p>The bacterium&nbsp;<em>Oceanicola batsensis</em>&nbsp;(NCMA B15&nbsp;from&nbsp;https://ncma.bigelow.org/) was purchased to serve as the primary bacterium for the heterotrophy experiment, but local heterotrophic bacteria were also cultured from the water near STRI. To isolate ambient bacteria, 250 mL of seawater from the site’s flow through system (that acquires water from a seagrass and hardbottom habitat approximately 100 m offshore) was filtered through a 0.22 µm polycarbonate filter. Sterile techniques were employed in a countertop, portable hood sterilized with 70% EtOH. The filter was placed into sterile 15 ml conical tubes with 8 ml of 0.2 µm filtered seawater and shaken gently. The filter was plated face down, and the water was streaked out onto the following agar preparations: 1.5% Difco marine broth 2216 with 0.2 µm unamended filtered sea water (FSW) and 1.5 % Difco marine broth 2216 with 0.7 µm FSW 1.5 % + 10 mM NH<sub>4</sub>Cl. These culturing techniques led to the formation of two colonies: JHB59-1.1 was a yellow, smooth colony isolated on 1.5% Difco marine broth 2216, and JHB59-3.2 were clear colonies isolated on 0.2 µm filtered seawater 1.5 % + 10 mM NH<sub>4</sub>Cl. Each of these, along with the purchased&nbsp;<em>O. batsensis</em>, were enriched in&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N for the heterotrophy experiments to measure sponge cell isotopic enrichment as a proxy for cell removal and assimilation.&nbsp;</p>
<p>&nbsp;A day prior to each experiment, colonies of&nbsp;<em>O. batsensis</em>, JHB59-1.1, and JHB59-3.2 were used to create starter cultures with 4 ml of marine broth. The evening prior to the experiment, the starter culture was added to 400 ml of marine broth, and the media was amended with&nbsp;<sup>13</sup>C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>&nbsp;(Glucose) (0.4. g L<sup>-1</sup>), NaH<sup>13</sup>CO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>),&nbsp;<sup>15</sup>NH<sub>4</sub>Cl (0.2 g L<sup>-1</sup>), and Na<sup>15</sup>NO<sub>3</sub>&nbsp;(0.1 g L<sup>-1</sup>). Dual carbon and nitrogen labels were used because one strain of&nbsp;<em>O. batsensis</em>&nbsp;does not utilize glucose and to ensure adequate&nbsp;<sup>15</sup>N enrichment. All isotopic tracer compounds had an atom % of the heavy atom of C and N (<sup>13</sup>C and&nbsp;<sup>15</sup>N) of at least 98%. Cultures were grown in an incubator at 29°C with rocking motion. On the morning of the experiment, the OD600 concentration was recorded for the overnight cultures using a spectrophotometer and then cells were pelleted by centrifugation in 50 ml Falcon tubes at 4500 x g for 10 min. Cells were resuspended in 12 ml of 0.7 µm filtered seawater for use in the experiment. Each day, the remainder of culture was stored in RNAlater and stored at -20 °C for future microbial 16S rDNA analysis.</p>
<p>The heterotrophy experiment was conducted over four consecutive days in a Latin square design with at least one replicate (one replicate on day #1 and two replicates in days two, three, and four) of each of the ten sponge species used per day. On the evening prior to the experiment, sponges were collected from the common garden reef and dark-adapted overnight in a common flow-through seawater tank. As above, at the start of the experiment, sponges were transferred to either 4 L or 6 L experimental containers filled with 0.7 µm (GFF) filtered seawater resting within a flow table with running seawater, and water circulation in experimental containers was maintained using a submersible pump for 120 min. Sponges were dosed with&nbsp;<sup>13</sup>C- and&nbsp;<sup>15</sup>N-enriched bacteria using a pipette at an initial average concentration of 3x10<sup>6&nbsp;</sup>microbial cells per ml. An initial 1 ml water sample was collected immediately at T = 0 for future flow cytometry analysis and this was repeated at 30 min intervals for T = 30, 60, 90, and 120 min. Data from the 90 min sampling were ultimately excluded from the study due to preservation issues. Samples were also collected from a control (without a sponge) tank dosed with the same concentration of bacteria as the sponge tanks each day; one control tank was included on day 1 and two controls tanks were included in days 2, 3, and 4 of the experiment. All water samples were added to a 2 ml screw top cryovial tube with 0.5% paraformaldehyde (final concentration), held at 4 °C for one hr, and then frozen at -20 °C. Water temperature was recorded every 30 min and ice packs were added to the surrounding water bath as needed. After 120 min of incubation with the enriched bacteria, flowing seawater was added to each tank via individual plastic tubing and tanks were flushed with ambient water for one hr to allow for the purging of enriched bacteria that might be transiting the sponge aquiferous system but had not yet been consumed. Following this, sponge tissue was rinsed in 0.7 µm filtered, unlabeled seawater, a small subsample was removed for microbiome analyses and preserved in 95% EtOH as outlined above, and the remaining tissue was wrapped in aluminum foil, and frozen at -20 °C for transport back to Charleston, SC. Sponges were later weighed to the nearest 0.001 g after thawing.&nbsp;</p>
<p>To determine the clearance rate for all ten sponge species, heterotrophic bacteria abundance was enumerated at each time point using flow cytometry. Cell counting was performed using a BD Biosciences Accuri C6 Plus Model flow cytometer at the Smithsonian Marine Station in Fort Pierce, Florida. The methods for staining heterotrophic bacteria with SYBR Green 1. Each day, fluoresce bright Plain YG 1.0 µm beads were added at 1.0 x 10<sup>6</sup>&nbsp;to 0.2 µm filtered seawater and run as a size standard, and a blank consisting of 1 ml TE (10 mM Tris-HCL, 1 mM EDTA) buffer and 10 µl 50x SYBR Green was also analyzed to differentiate signal from the TE buffer (distinct crescent shape output on the display) from the signal of experimental samples.</p>
<p>Each 1 ml experimental water sample was thawed and vortexed to ensure bacterial cells were fully resuspended. 100 µl of each sample was added to 900 µl of TE buffer and 10 µl of 50x SYBR Green in a new 1.5 ml centrifuge tube. This tube was inverted, incubated in a -20 °C freezer for 15 min in the dark, and run on the BD C6 flow cytometer at a flow rate of 35 μl min<sup>-1</sup>&nbsp;for 2 min. Samples were excited by a 488 nm laser and cell populations were detected using a 533/30 filter (FL-1), forward and side scatter with a threshold of 700 on FL-1. To reduce potential contamination across samples, the flow cytometer was backflushed, rinsed for one min with milliQ water at high flow rate (66 μl min<sup>-1</sup>), and the sample nozzle was wiped in between each sample. If a water sample (experimental or control) was analyzed more than once, then an average concentration of cells from all runs was ultimately used for a concentration value in the equations below.&nbsp;</p>
<p>The isolation of microbial and sponge cells from bulk sponge tissue followed methods adapted from Freeman et al. (2013). Sponge tissue was minced into small pieces using a sterile razor blade, added to 50 ml falcon tubes, and the sponge pieces were allowed to thaw for an hr at room temperature before being covered with ice-cold calcium-magnesium-free artificial seawater (CMFASW) amended with&nbsp;Ethylenediaminetetraacetic acid&nbsp;(EDTA; as in Freeman &amp; Thacker 2011). Samples were gently homogenized with a mortar and pestle 55x to dissociate the sponge and bacterial cells from the sponge matrix. This solution was filtered through 50 µm Nitex nylon mesh and lightly squeezed to retain undissociated cells and large spicules.</p>
<p>The resulting filtrate was poured into 50 ml centrifuge tubes and topped with CMFASW-EDTA for a final concentration of 50 ml. The filtrate for each sponge species was centrifuged at low speed to first isolate sponge cells and then high speeds to isolate microbial cells. To optimize sample purity, unique methods and speeds were used for each species (as in Freeman &amp; Thacker 2011, Freeman et al. 2013; Table S3). To isolate initial sponge cell fractions (containing some microbial cells), the above filtrate was centrifuged, and the supernatant after two to four low-speed rounds of centrifugation was decanted into a new 50 ml Falcon tube that was then centrifuged at 8000 RCF to obtain a microbial pellet (Table S3). Following this high-speed spin, the supernatant was poured off and the microbial pellet was stored at -20 °C. The initial pellets from low-speed centrifugation contained primarily sponge cells but needed to be purified again at lower speeds to remove microbial cells. Sponge pellets from consecutive low-speed centrifugation were resuspended in 15 ml of CMFASW-EDTA and combined. After each sponge pellet cleaning spin, the supernatant was discarded, and the cells were resuspended in 15 ml CMFASW-EDTA for the following spins. Purified sponge and microbial cell pellets were transferred to 2 ml micro-centrifuge tubes and centrifuged at 7200 RCF for 5 min at 4 °C. The supernatant was discarded, and the pellets were stored at -20 °C. Bulk sponge tissue was used for&nbsp;<em>Amphimedon compressa&nbsp;</em>because a pure sponge pellet could not be obtained due to cells rupturing during the dissociation process and because&nbsp;<em>A. compressa&nbsp;</em>has a very low abundance of microbial cells such that the microbial cell pellets lacked the biomass required for isotope analyses. Sponge and microbial fraction purity were verified using fluorescence and phase contrast microscopy at 400X (as in Freeman &amp; Thacker 2011).&nbsp;</p>
<p>The purified and frozen sponge and microbial fractions and subsamples of&nbsp;<em>A. compressa&nbsp;</em>tissue were lyophilized for 24 hr. Dried&nbsp;<em>A. compressa&nbsp;</em>was crushed with a mortar and pestle into a fine powder and transferred into 1.5 ml centrifuge tubes, and all dried pellets were placed in a 65 °C drying oven for 12 hr to remove any residual moisture. Samples were then acidified to remove any carbonate by exposure to 12 mol L<sup>-1</sup>&nbsp;HCl vapors for 12 hr in a sealed desiccator. Residual acid was removed following acidification by placing samples back into a 65 °C drying oven for 12 hr. Dried samples were weighed into tared tin capsules (Costech) to the nearest 0.001 mg (1.8 – 2.0 mg) using a microbalance.&nbsp;Crushed capsules were arranged into a 96-well plate and submitted for stable isotope analysis&nbsp;at the Smithsonian Institution OUSS/MCI Stable Isotope Mass Spectrometry Facility in&nbsp;Suitland, MD.&nbsp;Isotope ratios (δ<sup>13</sup>C and δ<sup>15</sup>N) of each fraction were measured on a Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV. All calculations of raw isotope values are performed with Isodat 3.0 software. Analytical precision at the Smithsonian Institution OUSS/MCI Stable Isotope Mass Spectrometry Facility is ± 0.2 ‰.&nbsp;</p>
<p>Sponge and microbial cell isotopic (<sup>15</sup>N and&nbsp;<sup>13</sup>C) enrichment was initially expressed in delta (δ) notation in permil units (<sup>0</sup>‰; in&nbsp;δ<sup>13</sup>C and δ<sup>15</sup>N values) but because initial natural abundance values were available for all species, enrichment for both experiments was ultimately expressed as change (Δ) in δ<sup>13</sup>C and δ<sup>15</sup>N (Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N value when compared to initial samples). These initial and experimental samples were matched for sponge genotype (same individual split among initial and experimental samples for&nbsp;<em>A. cauliformis</em>,&nbsp;<em>A. crassa</em>,&nbsp;<em>A. fulva</em>,&nbsp;<em>I. birotulata</em>,&nbsp;<em>M. laevis, N. erecta</em>,&nbsp;<em>V. rigida</em>, but not for&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>&nbsp;because these sponges cannot be easily and successfully partitioned into multiple individuals. Thus, for&nbsp;<em>I. bocatorensis</em>&nbsp;and&nbsp;<em>X. muta</em>, all seven initial (non-experimental) replicates were pooled to obtain an average natural abundance value for sponge and microbial cell fractions of each species. In addition, due to sample loss, there were only two initial&nbsp;<em>A. compressa</em>&nbsp;tissue samples; these two samples were used to obtain an initial average δ<sup>13</sup>C and δ<sup>15</sup>N value of holobiont tissue. These average initial δ<sup>13</sup>C and δ<sup>15</sup>N values were then subtracted from the δ<sup>13</sup>C and δ<sup>15</sup>N values of microbial and sponge cell fractions (or holobiont for&nbsp;<em>A. compressa</em>) from individual experimental replicates to obtain&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N values for&nbsp;<em>A. compressa</em>,&nbsp;<em>I. bocatorensis</em>, and&nbsp;<em>X.</em>&nbsp;<em>muta</em>.</p>
<p>Because&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the light treatment was derived from both autotrophic and heterotrophic microbial metabolism, but&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the dark treatment was only influenced by heterotrophic microbes, we isolated the role of autotrophic microbes in holobiont C and N metabolism in the light by subtracting&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment (calculated as&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N) in the dark treatment from total&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment (Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N) in the light treatment for each individual sponge sample. These values were ultimately expressed as % phototrophic and % heterotrophic metabolism according to the contribution of each pathway to total&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment in the light. Because&nbsp;<em>I. bocatorensis&nbsp;</em>and&nbsp;<em>X. muta</em>&nbsp;samples were not matched (the same genotype was not used in both light and dark experiments),&nbsp;<sup>13</sup>C and&nbsp;<sup>15</sup>N enrichment by phototrophic symbionts was measured by subtracting the average&nbsp;Δδ<sup>13</sup>C andΔδ<sup>15</sup>N values in the dark from average&nbsp;Δδ<sup>13</sup>C and&nbsp;Δδ<sup>15</sup>N values in the light.&nbsp;</p>
<p>Heterotrophic efficiency of particulate organic matter cell removal was calculated using clearance rate at an intermediate (60 min) and final (120 min) time point. Clearance rate (CR) represents the volume of water cleared of suspended particles per unit of time and is corrected by mass of the sponge according to the equation:</p>
<p>CR= (V/t<sub>x</sub>m) ln(C<em><sub>o</sub></em>/C<em><sub>t</sub></em>)</p>
<p>where V<sub>&nbsp;&nbsp;</sub>is the volume of the experimental tanks in ml (4000 ml or 6000 ml), t is time in hr (one or two hr), m is the wet mass of the sponge, C<sub>0</sub>&nbsp;is the starting concentration of bacteria in the tank, and C<sub>t</sub>&nbsp;is the concentration of bacteria at each time point (T = 60 min and T = 120 min). Because the concentration of labeled heterotrophic bacteria in control tanks varied minimally over the 120 min incubations with no significant decrease in cell number, clearance rates in experimental tanks were not corrected based on controls&nbsp;</p>
Specified by the Principal Investigator(s)
* split latitude & longitude into own column
* converted date to iso format (yyyy-mm-dd)
* Adjusted parameter names to comply with database requirements
Specified by BCO-DMO Data Managers
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
BD Biosciences Accuri C6 Plus Model
BD Biosciences Accuri C6 Plus Model
PI Supplied Instrument Name: BD Biosciences Accuri C6 Plus Model PI Supplied Instrument Description:Cell counting was performed using a BD Biosciences Accuri C6 Plus Model flow cytometer at the Smithsonian Marine Station in Fort Pierce, Florida. Instrument Name: Flow Cytometer Instrument Short Name:Flow Cytometer Instrument Description: Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
(from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm) Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB37/
Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV
Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV
PI Supplied Instrument Name: Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV PI Supplied Instrument Description:Isotope ratios (δ13C and δ15N) of each fraction were measured on a Thermo Delta V Advantage mass spectrometer in continuous flow mode coupled to an Elementar vario ISOTOPE Cube Elemental Analyzer (EA) via a Thermo Conflo IV. Instrument Name: Mass Spectrometer Instrument Short Name:Mass Spec Instrument Description: General term for instruments used to measure the mass-to-charge ratio of ions; generally used to find the composition of a sample by generating a mass spectrum representing the masses of sample components. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB16/
LI-COR LI-193 underwater spherical quantum sensor
LI-COR LI-193 underwater spherical quantum sensor
PI Supplied Instrument Name: LI-COR LI-193 underwater spherical quantum sensor PI Supplied Instrument Description:Irradiance was measured at multiple depths from collection sites around STRI using a LI-COR LI-193 underwater spherical quantum sensor. Instrument Name: Photosynthetically Available Radiation Sensor Instrument Short Name:PAR sensor Instrument Description: A PAR sensor measures photosynthetically available (or active) radiation. The sensor measures photon flux density (photons per second per square meter) within the visible wavelength range (typically 400 to 700 nanometers). PAR gives an indication of the total energy available to plants for photosynthesis. This instrument name is used when specific type, make and model are not known. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/122/