Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

This study was conducted at an artificial reef near Looe Key reef in the Florida Keys (USA), depth 10 meters. The artificial reef was removed after the experiment was completed.

To examine differences in nutrient profiles across sponge species, an artificial reef was constructed with 5 rows, with 10 cinder blocks per row, the rows were arranged in a semi-circle, and rebar was run through each individual row and secured at the middle and ends to ensure that the rows would not move over the duration of the experiment. Species (n = 10 replicates of 10 species) were placed on the rows in roughly a Latin-square design to spread out any environmental variable that may exist on the artificial reef across replicates of species. The depth of the reef was approximately 7 meters and immediately surrounding the reef was sand on all sides. The rows of sponges were placed so that they were as equal as possible in exposure to light and water flow. Two pieces of 5-millimeter (mm) polystyrene mesh were placed on top of each of the cinder blocks and secured with cable ties. Ten individuals of each of the 10 focal species (Aiolochroia crassa, Aplysina cauliformis, Aplysina fulva, Amphimedon compressa, Iotrochota birotulata, Ircinia felix, Niphates digitalis, Callyspongia aculeata, Verongula rigida, and Xestospongia muta) were selected and individuals were attached to the polystyrene mesh using cable ties. The sponges were allowed to acclimate for 4 months. The sponge species were selected based on prevalence in the Caribbean and included four low microbial abundance (LMA) species (Amphimedon compressa, Iotrochota birotulata, Niphates digitalis, Callyspongia aculeata) while the remaining six species are considered high microbial abundance (HMA) sponges.

In Situ Water collection:
Samples for all nutrient analyses were collected using a modular vacuum setup (VacuSIP) which was implemented and modified from Morganti et al. 2016. The VacuSIP included poly ether ether ketone (PEEK) tubing (used commonly in HPLC instruments) that was placed over the sponge osculum (sponge exhalent seawater) or near the sponge (inhalant seawater) and positioned using tripods. The tubing was connected via syringe needle to pressurized 250 milliliter (mL amber glass bottles with Teflon septate caps. The 250 mL glass vials were pre-combusted within days of sampling (6 hours at 450 °Celsius) and pressurized manually to -15 psi and this pressure increased slightly at the depth of the artificial reef. VacuSIP lines were acid rinsed in 10% HCl. Pumping was confirmed using fluorescent dye before each collection and the dye was allowed to clear before sampling. Tubing was then positioned directly above the pumping oscula for small sponges or inside the oscula close to the sponge for larger sponges. VacuSIP lines were then attached to the appropriate 250 mL bottles in the crates by sticking the needles at the end of the line into the septa of the bottle. Tubing for inhalant water collection was inserted into the appropriate 250 mL bottles. The apparatus contained fewer lines than 250 mL collection bottles, so the apparatus was set up to fill half of the bottles in the collection crate for 120 minutes, then the lines were moved to new bottles to fill the remaining bottles for 90 minutes. Once at the surface, the 250 mL bottles for each individual sample were combined into 2 liter (L) bottles, one for inhalant and one for exhalent water samples, that were labeled and then stored on ice in coolers until they were filtered.

Water Filtration:
A Cole-Palmer Masterflex L/S Intertek fitted with a Masterflex L/S easyload II head and Multichannel Pump Head Cartridges was used to filter the samples at a rate of 40 mL per minute through High-Performance Precision Pump Tubing, PharMed® BPT, L/S 15 with a 0.2 micrometer (µm) Supor filter into 1L acid-rinsed polycarbonate bottles that were covered with aluminum foil. The filters were archived for future microbiome analysis and the filtrate was stored in 40 mL amber vials with Teflon septa. Filtrate for dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) was acidified to ~pH 2 using 6N HCl and stored at 4°Celsius. Samples for DOC and TDN were sent to the UGA Stable Isotope Ecology Laboratory for analysis using a Shimadzu TOC-5000A Total Organic Carbon Analyzer. While DOC and TDN samples were collected, there was contamination in these samples from an unknown source and therefore, DOC and TDN were not used in any data analysis. Filtrate for fluorescent dissolved organic matter (fDOM) was not acidified and stored at 4°Celsius. The fDOM samples were shipped to a collaborator at the University of Hawaii (Dr. Craig Nelson) where they were stored until analysis. The investigator worked with the Nelson lab at UH to process and analyze the fDOM as samples described below.

fDOM Sample Processing:
Following the methods from Nelson et al. (2015), samples were analyzed with a Horiba Aqualog scanning fluorometer with 150 watts Xe excitation lamp, Peltier-cooled CCD emission detector, and simultaneous absorbance spectrometer. Quartz cuvettes of 1-centimeter (cm) diameter, which were DIW-leached and rinsed, were used to measure fluorescence. Samples were brought to room temperature while the Xe bulb warmed up.

Known problems or issues:
Not all replicates of each sponge were sampled. Dissolved Organic Carbon (DOC) and Total Dissolved Nitrogen (TDN) values are absent from the dataset due to contamination of samples.

Percent change in nutrients was calculated only for fDOM components, not for inorganic nutrients or for particulates captured on the GF/F filters.

Several samples were not analyzed for fDOM or for inorganic nutrients (broken or lost), and only a subset of samples were analyzed with the GF/F filters.

Fluorescent dissolved organic matter (fDOM) processing:
Excitation-emission matrices (EEMs), 3D contour plots of excitation and emission fluorescence, were measured from the 108 samples, starting with 4 DIW filled cuvettes as blanks at the beginning and end of the analysis. Scans were processed using a MATLAB script that use parallel factor analysis (PARAFAC) to identify peaks in the EEMs that correspond to previously characterized fluorescent components of humic-like and amino acid-like components (https://github.com/zquinlan/fDOMmatlab/script.md) (Coble, 1996; Nelson et al 2015).

Inorganic nutrient processing:
Filtrate (0.2 µm) was stored in acid-cleaned polypropylene bottles at -20° Celsius for inorganic nutrient analysis. Samples were shipped to Oregon State University (OSU) nutrient analysis laboratory. At OSU, Technicon AutoAnalyzer II™ components were used to measure phosphate and ammonium; and Alpkem RFA 300™ components were used for silicic acid, nitrate plus nitrite, and nitrite. Gordon et al. (1994) described analytical methods and data processing. The phosphate method was a modification of the molybdenum blue procedure of Bernhardt and Wilhelms (1967), in which phosphate is determined as reduced phosphomolybdic acid employing hydrazine as the reductant. The nitrate + nitrite analysis used the basic method of Armstrong et al. (1967), with modifications to improve the precision and ease of operation. Sulfanilamide and N-(1-Napthyl)ethylenediamine dihydrochloride react with nitrite to form a colored diazo compound. For the nitrate + nitrite analysis, nitrate was first reduced to nitrite using an OTCR and imidazole buffer as described by Patton (1983). Nitrite analysis was performed on a separate channel, omitting the cadmium reductor and the buffer. The method was based on that of Armstrong et al. (1967) as adapted by Atlas et al. (1971). Addition of an acidic molybdate reagent forms silicomolybdic acid which is then reduced by stannous chloride. This indophenol blue method is modified from ALPKEM RFA methodology which references Methods for Chemical Analysis of Water and Wastes, March 1984, EPA-600/4-79-020, "Nitrogen Ammonia", Method 350.1 (Colorimetric, Automated Phenate). All values are concentration in micromoles per liter of seawater.

Elemental analysis of particulates retained on glass fiber filters:
Combusted glass fiber filters (GF/F, 0.7 µm mesh, 13 mm diameter) were weighed prior to water filtration, then oven-dried at 60° Celsius for 24 hours before weighing again after water filtration. Dried filters were carefully packed into tin capsules (8 x 5 mm, EAconsumables), stored in a 96-well plate and shipped to the University of Georgia (UGA) Stable Isotope Ecology Laboratory (SIEL) for elemental analysis. Blank combusted filters were also weighed and added to the plate as background controls. At UGA, samples were analyzed for carbon and nitrogen content using a 2 Thermo Flash 1000 series elemental analyzer. Elemental standards included the National Institute of Standards and Technology (NIST) standard reference material 1570a. The percent carbon and nitrogen content were calculated for each sample and obtained from UGA SIEL.

- Imported original file named "fDOM_and_nutrients_combined_ForBCODMO_ArtificialReef2021_Fiore.xlsx" into the BCO-DMO system.
- Renamed fields to comply with BCO-DMO naming conventions (replaced spaces with underscores and removed or replaced other non-allowed characters).
- Rounded columns to 5 decimal places.
- Named the final file "906070_v1_artificial_reef_fdom.csv".

NSF Award Abstract:
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.

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.