Table of Contents

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

Coral reef cleaning stations were identified for survey at locations in Marathon, Florida (USA), St. Croix (US Virgin Islands), and Puerto Rico. SCUBA divers and snorkelers performed site surveys and fish observations at cleaning sites. All molecular samples were stored at -80 degrees Celsius or liquid nitrogen dry shipped until DNA extraction.

After observation, cleanerfish and damselfish were captured using individual hand nets and transferred to individual sealed plastic bags. Immediately upon capture, fish were taken to the lab and swabbed on both sides of the body with tubed sterile cotton swabs. Sampling was performed using gloves, and nets were submerged in a 30% bleach solution and rinsed with fresh water prior to each use.

At select coral cleaning stations, sterile clay hexagon tiles were deployed 1 meter above coral heads to develop biofilms. After 6-7 days, the tiles were recovered with minimal handling into Whirlpak bags and transported to the laboratory. The coral heads were also sampled for their mucus, and water was collected in 60-milliliter (mL) syringes from within 30 centimeters of the coral heads. Sterile swabs were used to collect samples from the tiles and coral mucus, and stored in 1.5 mL microcentrifuge tubes.

From select locations, water samples to examine macronutrients were collected from each station into acid-washed polypropylene bottles, frozen, and analyzed with a continuous segmented flow system at Oregon State University to resolve nitrate, nitrite, ammonium, phosphate, and silicate. Water for non-purgeable organic carbon (TOC) and organic nitrogen was collected from the stations into combusted, borosilicate EPA vials and acidified using 75 mL phosphoric acid. Samples were analyzed using a Shimadzu TOC-VCSH total organic carbon analyzer equipped with a TNM-1 module at Woods Hole Oceanographic Institution. To quantify abundances of Prochlorococcus, Synechococcus, picoeukaryotes, and unpigmented (heterotrophic) microorganisms, water (1.4 mL) was collected at the stations, preserved using paraformaldehyde, and frozen in liquid nitrogen vapors.

DNA extractions were performed on swabs of coral, damselfish, and cleanerfish mucus; swabs of tiles; and water filters. DNeasy PowerBiofilm DNA extraction kits (Qiagen, Germantown, MD, USA) were used with a modified protocol for the swab samples. Lysing reagents were added directly to the sample vial with the swab, vortexed, then the swab was re-oriented in the tube so it was cotton-side-up and was spun for 1 minute at 13,000 g to release the liquid absorbed by the swab. The liquid from the swab and the lysing reagents were added to the bead tube, and the manufacturer instructions were followed for the rest of the protocol. For water filters, the filter and lysing reagents were added directly to the bead tube, and then the manufacturer protocols were followed. DNA extraction controls (sterile hydroflock swabs, unused 0.22-micrometer (μm) filters, and unused bead tubes) were completed following the steps above.

FlowJo software (Tree Star, Inc.) was used for flow cytometry.

- Imported original file "bco-dmo_submission.xlsx" into the BCO-DMO system.
- Separated latitude and longitude values into separate columns where they were combined.
- Saved the final file as "906740_v1_sequence_sample_information.csv".

NSF Award Abstract:

Biodiversity in the ocean is influenced by interactions between disparate organisms which ultimately shape population, community, and ecosystem dynamics. Symbiotic interactions involving subsets of species can have disproportionate impacts on communities, reaching well beyond each interacting species. Coral reefs host some of the most iconic symbiotic interactions in nature and are host to the highest diversity of life on the planet. Cleaning symbiosis, wherein small fish or shrimp remove external parasites and associated microorganisms from specific clients, is common on coral reefs. Sites on the reef occupied by cleaners, or “cleaning stations”, attract a wide variety of fish species that engage in direct physical contact with the cleaner. These highly used territories are viewed both as “clinics of the sea”, where parasitized and sickly fish seek the service of cleaners, but also as potential “garbage dumps”, where unnecessary parasites and other microorganisms are removed. This project seeks to understand the role of cleaning symbiosis transferring microbes in coral reef environments. This research supports training for U.S. graduate students and for undergraduates from Arkansas State University, a primarily undergraduate institution that includes a large population of first-generation college students. These students participate in field site research and have opportunities to visit the Woods Hole Oceanographic Institution for broader exposure to ocean science and more specific laboratory training. The project strengthens international collaboration and further builds on the existing relationships between the team of scientists and resource managers, local divers, fishers, and boat operators, as well as K-12 schools and environmental education programs, and will therefore contribute to local economies. Outreach efforts include a film highlighting this research and publicly accessible narratives shared through press releases and an on-line magazine.

While the benefits of cleaning to reef ecosystem health have been extensively studied, the cleaning costs for cleaner species and the role of cleaning stations as potential sinks for microbial diversity and possibly even pathogens have never been assessed. Here, the researchers utilize the unique features of cleaning stations to understand transfer of bacterial and archaeal symbionts amongst fish and within coral reef environment. The study capitalizes on cleanerfish access to multiple variety of hosts or clients within stations to address new questions about how cleanerfish act as vectors to transfer microorganisms between hosts on a reef and if and how these microorganisms may play a broader functional role in reef resilience. Specifically, the project addresses the following hypotheses: 1) Cleanerfishes serve as keystone regulators of microbial communities, enhancing microbial community diversity and transferring key microbial species between clients, and 2) Cleanerfishes are a particularly important contributor to reef resilience, facilitating recovery of the microbiome following disturbance. The research team uses an integrative interdisciplinary approach involving field and laboratory observations and experiments, and molecular-based tools. The core research team includes experts in cleaning mutualisms, fish behavior, coral reef ecology, and microbial ecology. The proposed project aims to link behavior of individual organisms with ecosystem-level process.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.