In the past several decades Florida manatees (Trichechus manatus latirostris) have begun to expand their summer range into the northern Gulf of Mexico. Because this is a recent occurrence, not much is known about their habitat use and distribution in this region. Citizen-sourced sighting data suggests that Florida manatees frequent subembayments of Mobile Bay, Alabama, reaching a sighting peak in August. To assess the occurrence of manatees in this area environmental DNA surveys were used from winter (February 19-20) and summer (August 21-22) of 2018. At each of the 21 sites ranging from the mouth of Mobile Bay, to the Mobile-Tensaw Delta, Mobile-Tombigbee River, and Tensaw-Alabama River, 5 × 1 L water samples were collected. An additional water sample was collected from an ex situ experiment to gain a positive eDNA sample. This was done by adding feces and flesh from a deceased Florida manatee then collecting the water sample 30 minutes later. All water samples were vacuum-filtered, extracted for DNA, and run on Droplet Digital™ Polymerase Chain Reaction. A previously developed ddPCR assay was used to amplify a 69-base pair segment of the cytochrome b gene. The assay was able to detect 77.2 copies/µL of target DNA in the positive eDNA sample, 1.180 copies/µL in the 1:10 dilution, and 0.211 copies/µL in the 1:100 dilution of this sample. One summer field sample met one out of three criteria while another met two out of three criteria for a positive detection. There was evidence of contamination in several negative control samples that highlights the importance of negative controls in eDNA experiments
