International Journal of Marine Science, 2025, Vol.15, No.3, 118-129 http://www.aquapublisher.com/index.php/ijms 125 negatives or false positives. When the target species has extremely low density or only occasionally passes, its DNA concentration may be below the detection limit, thus missing detection. This is particularly prominent in large and rare fishes, requiring increased sensitivity by increasing water sample volume or enrichment means. Research by Bessey et al. (2020) shows that in tropical sea islands and reef areas, the volume of water sample has a significant impact on the number of species detected by eDNA: the number of fish OTUs detected by filtering 20 liters of seawater is about 30% higher than the 2 liter sample, and it is recommended to increase the sampling volume as much as possible to capture more DNA (Bessey et al., 2020). Secondly, environmental interference factors such as ocean currents and waves will have a dilution effect on the spatial distribution of eDNA. If the sampling coincides with strong water flow erosion, it may cause the local DNA to disperse rapidly. In this case, multiple parallel samples can be selected or repeated sampling in different tidal periods to reduce the influence of instantaneous factors. The presence of PCR inhibitory substances (such as humic acid, heavy metals, etc.) in seawater may also affect sensitivity (Pritam Banerjee et al., 2022). For waters affected by river runoff in the offshore Hainan, additional purification steps are required after DNA extraction, or inhibitor-resistant enzymes are used during amplification to overcome potential PCR inhibition. Another problem is the fragmentation of environmental DNA: DNA degradation is faster in high temperature seawater, and the fragments may be too short to make it impossible to amplify barcode fragments of the expected length. Solutions include the use of shorter amplification fragments (such as very short barcode mini-barcode) or the addition of storage solution immediately after sample collection to reduce the degradation rate. 6.2 The impact of imperfect species identification database A core link in environmental DNA analysis is the species annotation of sequences, which relies on a complete database of reference sequences. The current challenge is that species endemic to certain regions or not sequenced lack corresponding sequences in the database, resulting in the inability to accurately identify eDNA sequences. This is particularly prominent in biodiversity hotspots such as Hainan Island. Some endemic fish in the South China Sea have not yet determined the DNA barcode sequence, so eDNA can only identify the level of family or genus when detecting its DNA, and the species name cannot be confirmed. Jaquier et al. (2024) found through global analysis that there is a clear "species vacancy" phenomenon in the fish DNA database: about 20% to 30% of known fish species lack 12S barcode sequences. The imperfection of this database directly affects the accuracy of eDNA application in species identification. To solve this problem, it is necessary to carry out regional DNA reference library construction work: collect tissue samples of common and endemic fish in Hainan Island and the South China Sea, determine standard barcode sequences and upload them to the database. Continuing to improve the sequence data of local species will greatly improve the success rate and accuracy of eDNA species allocation (Read et al., 2022). Another strategy is to adopt multigene loci to improve identification resolution. For example, sequencing the mitochondrial COI gene (often used in DNA barcode) and the 12S gene simultaneously will help distinguish relatives. However, this also requires the completeness of the COI database. 6.3 Construction of standardized processes and regional adaptability To truly apply eDNA technology to routine monitoring, the problem of standardization and local adaptation needs to be solved. At present, different research teams may have differences in primer selection, filtration volume, data analysis threshold, etc., resulting in limited comparability of the results. In order to serve the management of Hainan's marine ecology, it is necessary to establish a set of eDNA operation specifications suitable for the region. This includes: formulating standard operating procedures (SOPs), which should be based on the optimization results of previous trials and are in line with international common practices to make the data have horizontal comparison significance. Secondly, establish quality control and data interpretation standards. For example, set the sequence reading threshold, and species records below a certain reading should be treated with caution; it is clear that no fish sequence was detected by negative controls as a qualified experiment, otherwise the entire batch of data will be invalid. Again, in terms of regional adaptability, adjustments can be made to the particularity of Hainan sea area (Figure 2). For example, if the water temperature along Hainan coast is high, you can consider shortening the interval between sampling and experiments, or refrigerating the water samples on site to protect DNA integrity. A local reference database update plan for Hainan should also be formulated, and new species
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