Detecting Mediterranean White Sharks with Environmental DNA

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back to 1860 are being used to estimate population trajectories throughout the entire region. Though the population size is unknown, remaining individuals are thought to be primarily restricted to a handful of hotspots deemed important for their reproduction and foraging. One of these hypothesized hotspots is the Sicilian Channel, which accounts for 19% of total historical sightings.
White sharks shed genetic material (skin and feces) into their environment. Non-invasive molecular methods for determining the presence of organisms in nature, such as detecting environmental DNA (eDNA) from water samples, offer a developing but powerful technique for studying cryptic or rare species. In the Mediterranean Sea, where white shark catches and sightings are limited and unpredictable, the analysis of genetic material shed from white sharks provides novel insights into their distribution and abundance, thus aiding traditional surveying methods based on encountering individuals.
Here, we describe a survey conducted in the Sicilian Channel to test and refine methods for detecting white shark eDNA and combine historical aggregations with movement simulations of eDNA molecules in seawater to interpret white shark distributions and set goals for future monitoring efforts.

WHITE SHARK EDNA SURVEY
In June 2021, we collected environmental water samples from 16 sites within the Sicilian Channel (Figures 1 and 2).
We filtered the samples to capture any cells and freeflowing eDNA that may be present. We amplified a unique cytochrome B gene fragment (CYTB) found in the mitochondria of white shark cells (Lafferty et al., 2018). We then observed the CYTB gene fragment on gel electrophoresis to confirm that the sample contained white shark DNA. DNA molecules are subjected to environmental factors such as water currents and chemistry, tides, temperature, and sunlight once they are shed by white sharks.
By modeling the trajectory of detected eDNA molecules,  FIGURE 2. Seawater was sampled and analyzed for genetic material. (a) Two to four liters of seawater (depending on available labor and processing time) were collected in duplicates at depths from 10 m to 100 m with a 5L Niskin bottle and (b) filtered using 0.2 µm pore size filter paper. (c) eDNA was then extracted and a polymerase chain reaction (PCR) was performed to amplify the white sharkspecific CYTB gene. At the PCR step, sterile water was used as a negative control to imitate a sample with zero concentration of white shark eDNA, thereby providing a frame of reference for amplifying the CYTB gene fragment. (d) White shark genetic material was retrieved from native seawater and eDNA was visualized on a gel electrophoresis machine. (e) At the Virginia Tech Genomics Sequencing Center, sampled genetic material was visualized on a TapeStation machine with a positive control (genomic DNA) derived from white shark tissue. To prevent contamination, genomic DNA was not used at sea. Steps a-d were conducted on the boat in real time and thus were helpful for identifying sampling sites where white sharks might have been present recently in order to focus effort on observing individuals to further confirm their presence.
Step e was conducted following the expedition and in conjunction with DNA sequencing. The d-panel illustration was adapted from Chavez et al. (2021).

INVOLVING THE PUBLIC IN OUR WORK
Our approach is effective and scalable for clarifying spatio- surface water and preserving eDNA to be shipped back to our laboratory for processing. To teach citizens about the best sampling practices, we created a short instructional document and a step-by-step video to help them collect water, sterilize the equipment, and work area with diluted bleach; use latex gloves; and preserve eDNA. As the network expands, kits should be upgraded to sample at depth to help reveal the population distribution of white sharks in the Mediterranean.
In summary, detecting the DNA of Critically Endangered white shark individuals allowed us to extrapolate their spatiotemporal presence and develop a surveying strategy in a data-poor region. Expanded systematic eDNA monitoring is a promising tool for revealing the otherwise cryptic behavior of white sharks in the region. This will be crucial for developing conservation and management policies to protect the last strongholds of Mediterranean white sharks.