4.3. Cost and effort
The additional cost of adding eDNA analyses to an existing water quality program is significantly less than the cost of an eDNA-specific sampling, primarily due to savings in field time and equipment. If samples can be collected and filtered in conjunction with other water quality samples that require filtration (nutrients, for example), the estimated additional resources required for collecting samples, filtering, and preparing for shipment is $5-$20 and 15-60 minutes of staff time per sample. If eDNA samples are collected separately, but leverage existing site personnel and equipment, the per sample cost is $6-$30 and 1-2.5 hours. Costs vary widely depending on boat charges, distance to sites, and number of sites sampled in a day. The cost of lab and bioinformatics analyses varies depending on type of analyses, number of samples, and available resources. Results may be returned as raw sequence reads, or may be processed to identify organisms. When raw data is returned the user must have access to both the skill and computational capacity to perform bioinformatics, which may be a significant barrier for some resource managers. Analysis of eDNA samples is increasingly available from commercial vendors with costs ranging from $75-$150 per sample for established metabarcoding analyses. Costs are higher for projects with small sample batches, project advice, additional analyses etc.
5. CONCLUSIONS
eDNA sampling is relatively easy to implement, and can be applied in areas which are difficult to reach with traditional surveys (sensitive habitats, hard to access locations). Sample collection can be added to existing water quality monitoring programs at less cost than implementing a new survey, and may increase detection of some species. Due to the relatively short time required for sampling, higher resolution sampling (temporally or spatially) can be conducted to gain more information about species at the sites. Sample extracts can be re-used for multiple target species or broad biodiversity metrics, and can be archived for future analyses. While we have not discussed genetic studies here, emerging methods in understanding sequence variants and microsatellites are moving towards establishing these methods (e.g. Dugal et al., 2022; Weitemier et al., 2021). However, there are caveats to implementing an eDNA monitoring program: Interpretation of results can be ambiguous, and incomplete sequence data, transport of DNA, and misidentification of species can lead to errors.
eDNA fish surveys are a potentially powerful addition to standardized monitoring programs, but have limitations. In Great Bay, extensive eDNA sampling detected all of the fish likely present, including species that are difficult to catch in seine nets. In Heʻeia, there was general agreement with the presence of non-native, freshwater fish found in estuarine sites through comparison of eDNA with cast net and visual sampling and eDNA allowed the detection of specific native species that are cryptic or difficult to locate. However, in the highly turbid Apalachicola estuary the eDNA sampling and analysis conducted in this study underperformed traditional sampling and missed many species likely to be present. At many estuarine locations, more intensive sampling, additional lab processing, application of more or different primers, and augmentation of the sequence database using place-specific local fish voucher specimen will yield better results. In this study we deliberately apply the same methods at all sites, rather than optimizing at each location so that we can understand the value of a broad application of a set of common methods. Managers should consider the individual goals and needs of their program to determine if this approach is appropriate and useful. We strongly recommend that resource managers considering eDNA applications become familiar with the methods, consult experts with local knowledge about fish species, and work with sequencing centers or partners that can support accurate interpretation and troubleshooting of results.
The results of this study support previous work finding that eDNA sampling is a potentially powerful tool for fish assessments in estuarine systems. Results will be improved by collecting replicate samples, focusing sampling in locations or seasons of interest, and by collecting local voucher specimens for closely related species.
AKNOWLEDGEMENTS
We are grateful to students, interns and collaborators at each site who assisted in sample collection: Claire Gottsegen, Diana Lopera, Nakoa Goo, Hoaka Thomas, Aka Beebe, Becca Lensing,
Gus Robertson, Kim Falinski, Anthony Olegario. This work was supported by the National Estuarine Research Reserve System (NERRS) Science Collaborative which is funded by the National Oceanic and Atmospheric Administration (NOAA) and managed by the University of Michigan Water Center (NAI4NOS4190145).
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DATA ACCESSIBILITY: All raw data and associated metadata is in the processes of being uploaded to NCBI SRA, under bioproject PRJNA667067.
BENEFITS GENERATED: Benefits from this research accrue from the sharing of our data and results on public databases and this open access paper as described above.
AUTHOR CONTRIBUTIONS: A.W, C.P., J.S.G., B.Y., and W.K.T designed the study. J.S.G., C.P., J.G., M.L., S.S., Y.R., collected samples. D.T. conducted data analyses, W.K.T oversaw lab analyses. A.W. wrote the manuscript with review from all authors.
CONFLICT OF INTEREST: The authors declare no conflict of interest