International Journal of Marine Science, 2025, Vol.15, No.3, 118-129 http://www.aquapublisher.com/index.php/ijms 120 water is collected per sample point and eDNA is enriched by on-site filtration (Kristin et al., 2020). Filtration is a key step in capturing eDNA. Commonly used filter membrane materials include cellulose acetate, nylon, etc., with a pore size of generally 0.45 μm or 0.22 μm, which can intercept free DNA and cell debris. The material and pore size of the filter membrane will affect DNA recovery. Hinlo et al. (2017) compared different filtration and precipitation methods and found that filtration method can more effectively enrich eDNA in water, while filter membrane types (such as nitrofibrous filter vs. polycarbonate filter membrane) also have an impact on DNA yield (Hinlo et al., 2017). Generally speaking, 0.45 μm fiber filter membranes are often used in seawater samples because they take into account the faster filtration speed and higher DNA capture efficiency. After filtration is completed, the filter membrane needs to be stored immediately. Common methods include placing the filter membrane in anhydrous ethanol, or storing it at low temperature after adding DNA protection reagents, or quick-freezing of the filter membrane on site. These measures are designed to inhibit the DNA enzyme activity of microorganisms on the filter membrane and prevent eDNA degradation. During transportation and laboratory preservation, the filter membrane should be kept in a freezing environment of -20 °C or even -80 °C until DNA is extracted (Troth et al., 2020). Studies have shown that timely cryopreservation can significantly improve the success rate and yield of subsequent DNA extraction. 2.3 DNA extraction, amplification and sequence analysis process Extraction of DNA from filter membranes is the second step in eDNA analysis. Currently commonly used DNA extraction kits (such as Qiagen DNeasy, etc.) have been proven to be effective in recovering environmental DNA from filter membranes. During the extraction process, the filter membrane is usually cut into pieces and placed in the lysate, and total DNA is obtained by digestion and elution of protease K. Because the total DNA concentration in the water sample is very low and the target DNA fragment length is short (usually 100~300 bp), specific gene fragments need to be amplified by polymerase chain reaction (PCR) to improve the detectability of the DNA of the target species. The MiFish primers developed by Miya et al. are a set of primers that are widely used in fish eDNA metabarcodes and can cover the vast majority of bone and cartilage fish species (Miya et al., 2015). These primers amplify 12S gene fragments about 170 bp long, suitable for high-throughput sequencing platforms. The amplification reaction requires strict prevention of contamination, and usually a negative control (no DNA template) and a positive control (DNA of known sequences) are set up to monitor PCR quality. After successful amplification, the product was purified and a sequencing library was constructed, and sent to a high-throughput sequencer to obtain massive sequence data. Next is bioinformatics analysis: the output sequences are quality-controlled and deredundantly processed, clustered into operational taxonos (OTUs) or resolved into amplicon sequence variants (ASVs), and then aligned with the reference database for species annotation. After data comparison, information such as species composition and sequence read abundance in the sample can be obtained. Finally, the species list and readings are combined with statistical analysis to compare community differences at different points or different time periods, and the species diversity index can be evaluated. 3 Development of eDNA Technology in Aquatic Ecology Research 3.1 Main research results and typical cases 3.1.1 EDNA monitoring of fish diversity in offshore coral reefs in Hawaii The coral reef ecosystems around the Hawaiian Islands have abundant species and are highly endemic, and have always been one of the hot spots for marine biodiversity research. Although there are relatively limited research on eDNA published in Hawaiian coral reef fish, relevant attempts have shown that the technology is feasible in the region. Marine biologists conducted pilot studies in a protected area near Oahu Island in Hawaii. Through eDNA analysis, a large number of coral reef fish DNA signals, including butterfly fish, cannonball fish, etc., were detected, and were highly consistent with the list of species surveyed by traditional underwater visual surveys. Another team study of NOAA plans to conduct a comprehensive application of eDNA on the west coast of Hawaii's Great Island in 2022. The program combines eDNA with three-dimensional reef imaging and artificial visual census to compare the pros and cons of different methods. These efforts herald the hope of eDNA to achieve integrated monitoring of the entire coral reef ecosystem “from microorganisms to large fish” in waters such as Hawaii (Friedlander et al., 2022). Although there are not many specific eDNA research results in Hawaii
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