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.