The aim of our research is the survey on biological communities at high seas using commercial vessel as a platform for the collection of environmental DNA samples. In our specific case, the focus is on cetaecans. Try to identify offshore area, ecologically strategic for their protection. While the acquisition of EDNA sample is extremely easy, just collecting seawater sample, and although the analytical technology is constantly progressing, the real challenge is how to reach the environments to be monitored, especially when these are difficult to access, such as in the case of offshore waters of deep waters. While EDNA is frequently becoming the elective technique for the study of the marine environment, protocols that allow for easy, and reproducible large volume sample processing in the field are missing. As EDNA degrades in a time span of hours, it is pivotal to filter and store seawater EDNA samples after collection to ensure maximal retrieval and efficiency. The presented protocol relies fully on operating ferry boats for data collection. Ferry based monitoring offers several advantages, such as the repeatability, roads are constant and run all year round, allowing seasonal monitoring, easy access to offshore waters, carrying out an emission free sample collection, making sampling possible also during nighttime hours, and finally reducing drastically the sampling costs. Using this protocol, we collected and filtered large numbers of samples, reaching remote and offshore areas, allowing for increased spatial resolution for our research. This is especially relevant as we target marine organisms that live in open sea, namely marine mammals. Has the information obtained from their EDNA could shed light on their distribution, habitat preference, and ultimately conservation status.
[Narrator] To begin, note down the current or average cruising speed of the ferry on the route to estimate the extension of the sampling stretch. After sanitizing the filtering surface, slightly open the sample collection tap for the duration of the cruise. Use the continuous flow of sea water to rinse the collection pipe with local water disposing of the excess water in the bilge. Use a permanent marker to label the BIB with the required details. Remove the ceiling film from the BIB opening, and fill it with approximately 13 liters of seawater. When the BIB is nearly full, remove the ceiling film from the lid, leaving the film around the tap intact. After sealing the BIB tightly, note the exact time when sample collection is completed, and record the total duration of the sample collection. Write the completion time on the BIB using a permanent marker, and transfer the information onto the sample collection and filtration form. Place the filled BIB in the storage room or filtration area. To prepare the filtering station, connect the vacuum pump to the suction nozzle of the vacuum flask, and place the stopper with the support on the neck of the flask to accommodate the filtration cylinder. Prepare all necessary materials, and assemble the filtering system in the designated space for filtration. Use a new cylinder for each station, and a new pair of single use tweezers or sterilized tweezers. To minimize the contamination risk, place an insulating sleeve such as a plastic bag between the tap and the filtration cylinder to isolate the water flow. Activate the vacuum pump, and start the timer to record the filtration time. Pass up to four liters of water per filter, ensuring the cylinder remains full throughout the process to avoid air entry. When the filtered water reaches the four liter mark, turn off the vacuum pump, and stop the timer immediately. Record the filtration time in the data log sheet. Then use tweezers to carefully recover the first filter or filter a from the cylinder, avoiding any damage. Folded it in half with the side retaining biological material folded inward, and wrap it in aluminum foil. Label the foil with the sample number, and filter replicate ID before storing it in the freezer. Mount a new filter inside the filtration cylinder for the next round of filtration, and empty the flask. After filtration. Store all filters between minus four degrees Celsius, and minus 20 degrees Celsius until further laboratory processing. Just before disembarkation, retrieve all samples, and place them in low temperature transportable coolers for transport. Marine environmental DNA collection, and analysis using meta bar coating, identified multiple vertebrate species including Tilefishes Alas Brinks, and cetaceans along the sampled route, enabling the reconstruction of vertebrate community composition, and trophic structure.
