Sample collection

For each captured juvenile turtle body size was determined, measured as straight carapace length (SCL), from the nuchal notch at the anterior edge of the carapace to the posterior tip of the supracaudal scutes. Following measurement, cloacal swabs were taken. Although cloacal swabs are often used as a proxy for fecal samples to characterize the animal’s gut flora [13], swabs from the turtles sampled for this study are likely influenced by the surrounding environment due to the turtle’s constant contact with water in the marine setting. To minimize this environmental influence and the potential for contamination from surrounding skin microflora, the exterior edge of the cloaca and tail of each turtle were sterilized with a 70% isopropyl alcohol swab after being placed on a table (wiped with 70% isopropyl alcohol) ventral side-up. Swabbing was carried out with sterile ultrafine (smaller pelagic turtles) or standard (larger neritic turtles) Hydraflock^® swabs (Puritan Medical, Guilford, Maine, USA). The swabs were dipped into sterile water before being inserted approximately the length of the swab tip (~ 2 cm) into the cloaca and rotated 2–3 times before removal. The swab tip was then cut from the handle using sterilized scissors and stored in RNAlater^® solution (ThermoFisher Scientific, Waltham, Massachusetts, USA), which allows extended storage at temperatures above -20°C. Duplicate swabs were taken from each turtle, and then all swab samples were stored at ~4°C in solution for up to five days before being transferred to -20°C for long term storage until thawing for DNA extraction.

For the pelagic juvenile turtle sampling (N = 9), capture methods followed those previously described [9]. During time onboard (approximately 1-hour), captured turtles were kept on a sterilized surface for opportunistic collection of fecal samples; however, only one individual produced a fecal sample. This sample was collected aseptically in a sterile Eppendorf tube containing RNAlater^® and stored as described above. Capture techniques for neritic juveniles from the bays (N = 3) and beachfront (N = 6) were location-dependent. In SJB and SAB, the capture methods were followed as previously reported [14]. Due to poor weather conditions and marine mammal sightings, our ability to consistently use the tangle net was limited and therefore one turtle was caught opportunistically with a dip net following a sighting in SJB. Sampling in the beachfront habitat on SRI used both utility task and all-terrain vehicles, which were driven along the beach while observers looked for juvenile green turtles in the surf zone. If a turtle was spotted, a 10 m tangle net was used to seine in front of the individual, while the remaining team members approached from behind the turtle to coerce it into the tangle net using dip nets. Five individuals were captured on SRI, and one individual was recaptured one week later for a second sample.

All animal sampling was carried out in strict accordance with the Purdue Animal Care and Use Committee (PACUC) policies and guidelines, and under PACUC approved protocol #1502001194. All captured turtles were released shortly after sampling was complete.