Unveiling the power of environmental DNA: a review of eDNA application to monitoring threatened fish in Indonesian Waters

. This study examines environmental DNA (eDNA) techniques to detect and monitor threatened fish species in Indonesian waters. By analyzing 17 selected documents out of 284 papers and 310 metadata files, the study identified 30 Indonesian threatened fish species through eDNA technology which only assessed 8.5% of 352 threatened fish species in Indonesian waters based on Fishbase data, particularly in families such as Osphronemidae, Melanotaeniidae, Dasyatidae, Carcharhinidae, and Gobiidae. The paper discusses estimating fish abundance using eDNA analysis, including sample collection, eDNA concentration analysis, correlation with fish biomass, and field application. Challenges and limitations in estimating fish abundance through eDNA are addressed, such as misinterpretation, environmental factors, eDNA persistence, and sampling bias. The findings highlighted the potential of eDNA as a valuable tool for detecting and monitoring threatened fish species in Indonesian waters. However, further research and conservation efforts are needed in this area. By addressing the identified challenges and limitations, a better understanding of threatened fish populations can be gained, leading to effective conservation strategies and the long-term survival of these species in Indonesian waters.


Introduction
Indonesia is home to different types of fish, comprising 1,259 species living in the freshwater and 3,682 species inhabiting marine ecosystems [1].However, some Indonesian native fishes risk becoming extinct due to human activities such as overfishing, habitat degradation, habitat fragmentation, water pollution, and the introduction of invasive species.According to the Indonesian Ministry of Marine Affairs and Fisheries Decree No.1/2021, 20 fish species have been under full protection.
Implementing management and conservation efforts that consider biota and habitat management is important.Monitoring fish distribution, abundance, and their migration patterns in the wild is vital for determining the fishing ground, fishing season, and conservation areas.However, traditional techniques such as electric fishing and snorkeling, BRUV, along with electronic tagging using PIT, radio, and acoustic telemetry, are both costly and labor-intensive when it comes to evaluating the presence, abundance, and migration of fish [2][3][4][5].Moreover, the standard monitoring procedure requires taxonomic experts, and the sampling process may damage the habitat or harm the species because the species needs to be caught [6].
Cryptic and rare species detection in any environment is challenging, especially in aquatic environments.Despite the low detection probabilities, it is essential to continue monitoring and researching these rare species to understand their behavior better and protect them from threats.Moreover, fish abundance monitoring in threatened populations is important for conservation planning, monitoring population trends, evaluating conservation actions, understanding threats, and establishing conservation priorities.
The use of environmental DNA (eDNA) has been found to be effective in many fish studies, including for the detection of rare or threatened species [7][8][9][10][11].eDNA assay has proven to have higher detection power and lower costs than traditional techniques.Studies have shown that eDNA assay can contribute to fish management, and its performance complements the traditional methods [12].Many eDNA studies have reported the relationships between eDNA concentration and fish relative abundance [13][14][15][16][17]. Estimating the relative abundance through eDNA-based methods is essential in providing critical information.Although inconsistent results among the studies, including high variability in eDNA concentration between the samples, eDNA assay is still a promising tool for estimating fish abundance/biomass, which could be useful in the future for conservation efforts, managing fisheries, assessing ecosystems, and conducting research on biodiversity [18].
This paper provides an overview of the development and application of eDNA approaches for detecting and monitoring threatened fish in Indonesian waters.The exact number of threatened fish inhabiting Indonesian waters and eDNA studies performed in detecting and monitoring threatened fish are discussed in this paper.We will also explore the progress of eDNA-based abundance studies in fish species, including threatened fish, and the challenges of applying this method in the field.

Methods
The study utilized the Fishbase website [1] to examine various categories of fish species in Indonesia, including threatened, endemic, aquarium trade, and commercially important species.A systematic review approach was conducted to gather information specifically related to threatened fish species in Indonesia detected through eDNA analysis.The review also encompassed studies on eDNA for fish abundance estimation, the development of eDNA techniques for assessing threatened fish abundance, and the obstacles encountered in estimating fish abundance through eDNA using narrative literature reviews.Data and publications relevant to the research were collected from the Scopus database until May 28, 2023, using specific keywords (Table 1).These keywords included combinations related to Indonesian threatened fish detected by eDNA, eDNA studies and development for fish abundance, and various obstacles in estimating fish abundance using eDNA.Additionally, numerous other publications in English or Bahasa were obtained from Google Scholar, focusing on the usage of eDNA for Indonesian threatened fish.A total of 284 papers, which include journal and proceeding articles with full-text accessibility, and 310 papers available as metadata information (e.g., title and abstract) were identified.To eliminate duplicates, data and publications in the formats ".bib" and ".pdf" were removed using Mendeley Desktop.The analysis and review process involved applying filters based on keywords related to endangered and threatened fish, fish abundance, and obstacles in estimation.The systematic review included 17 papers focusing on endangered fish in Indonesia.Additionally, 49 papers were analyzed to gather information on endangered fish worldwide, and 11 papers specifically discussed the primers used in eDNA analysis.The data and information extracted from these papers were tabulated and processed using Microsoft Excel.The findings are presented descriptively through narrative explanations, tables, and figures, aiming to understand the topic comprehensively.

Results and Discussion
The findings highlight the significant number of threatened fish species in Indonesia, with 352 species identified as threatened.These species are categorized as 47% Vulnerable (VU), 34% Endangered (EN), and 19% Critically Endangered (CR).Furthermore, our findings identify several families with many threatened species, including Osphronemidae, Melanotaeniidae, Dasyatidae, Carcharhinidae, and Gobiidae.Our study also identifies 44 endemic fish species among the threatened species (Table 2).In the context of Indonesian threatened fish species, eDNA has detected 30 species (Table 3), which accounts for 8.5% of the total threatened fish species identified (Table 2).Table 3 and Figure 1 provide further details on the specific fish species detected using eDNA in Indonesia.Additionally, Table 4 presents information on the primers used in eDNA studies in Table 3.It is important to note that eDNA techniques have also been utilized globally to detect over 60 threatened fish species (Table 5), highlighting its widespread applicability.Regarding the taxonomic distribution of detected threatened fish species in Indonesia, Chondrichthyes (cartilaginous fishes) account for more than 70% of the findings based on group classification (Table 3; Figure 1).
w mICOlintFadapt COI F-TCGTCGGCAGCGTCAGATGTGTATAAGA GACAGGGWACWGGWTGAACWGTWTAY [42] *SharkCOI: SharkCOI-MINIR, 588 F: 588 F-limbatus-NADH2, 776 R: 776 R-limbatus-NADH2, 12S r.: 12S rRNA Opsariichthys uncirostris uncirostris [8] In Indonesia, Fishbase has identified over 300 threatened fish species classified as Vulnerable (VU), Endangered (EN), or Critically Endangered (CR), along with 44 species that are endemic to the region (Table 1).Interestingly, based on available information, Fishbase does not classify Near Threatened (NT) species, such as Anguilla, as threatened fish.However, it is concerning to note that many fish species in Indonesia face potential threats despite not being officially classified as threatened by Fishbase.Among these are 137 endemic species, 871 species involved in the aquarium trade, and 688 commercially important species [1].These species are at risk due to overexploitation, limited distribution, and habitat fragmentation or degradation.Furthermore, certain species like Chromobotia macracanthus and Datnioides microlepis, despite their commercial importance and use in the aquarium trade, still hold a Least Concern (LC) status.This highlights the importance of continuously assessing the conservation status of these species to ensure appropriate conservation measures are in place.The cumulative representation of threatened species in Indonesia is significantly attributed to the Osphronemidae, Melanotaeniidae, Dasyatidae, Carcharhinidae, and Gobiidae families, accounting for 41% of the total number of threatened species.Understanding the threats these families face is crucial for implementing targeted conservation efforts to safeguard their long-term survival and preserve Indonesia's rich aquatic biodiversity.This emphasizes the importance of comprehensive conservation strategies and continuous monitoring and assessment of fish species to address the threats they face effectively.
Environmental DNA technology has been instrumental in identifying threatened species, including in Indonesia.This highlights the significance of eDNA in detecting threatened species, particularly within this taxonomic group.In Indonesia, eDNA applications have been widely employed.Most eDNA studies in Indonesia use the eDNA metabarcoding approach, a technique for identifying multiple species from a single DNA sample, for various purposes, including species detection, distribution mapping, community structure assessment, and biodiversity evaluation.However, it is worth noting that the current focus of these studies in Indonesia has not been on estimating fish abundance (Table 3).Several primers have been used for detecting fish eDNA from the marine and freshwater.However, the primers used by [91] to detect freshwater fishes are not listed in this review because the primers used in their publication are species-specific primers to detect a frog (Rana catesbeiana), which were developed by [92].
The species-specific eDNA approach using qPCR is commonly used to detect single species and quantify the eDNA concentration.Fish abundance studies typically rely on presenting the number of DNA copies or eDNA concentrations [19,32] without conducting laboratory investigations to assess fish density or biomass [13,14,93].The evaluation of eDNA concentration and abundance of several freshwater species in natural habitats was accomplished by [14,18] through the application of species-specific qPCR-based eDNA assay.[13] expanded on the development of eDNA to quantify fish biomass/abundance in environments in 2012.They highlighted the various stages of developing eDNA technology for this purpose.In 2022, [94] published a comprehensive evaluation of 63 eDNA research published between 2012 and 2020.According to their evaluation, 90% of this research found a positive correlation between eDNA concentration and the target species' abundance and/or biomass.However, several biotic and environmental factors influence eDNA concentration in the field; therefore, careful interpretation should be conducted when estimating fish abundance through eDNA concentration.In pursuing advancing eDNA techniques, [95] have presented a conceptual framework for quantifying the fish abundance and biomass using eDNA methods.
Despite its limitations and bias, eDNA still has the potential to be used to estimate fish population abundance by further developing and validating the eDNA techniques for accurate estimation of fish abundance, including in Indonesian waters.Our review found that limited studies focus on using environmental DNA to assess fish abundance in Indonesian waters.Specifically, we identified two notable studies that have been conducted on the eDNA of Anguilla bicolor and Balantiocheilos melanopterus.eDNA species-specific approach using qPCR assay for fish detection is developed in stages, including laboratory experiments, pond trials, and field surveys.These steps are also critical for improving and verifying the use of eDNA to estimate fish abundance.The progress of eDNA studies by Larashati et al. and Kusumah et al. are described in Table 6.Estimating fish abundance using eDNA approaches can be accompanied by several obstacles and limitations [94].Some of these challenges are: (a) Careful interpretation.Interpreting the relationship between eDNA concentration and fish abundance or biomass requires careful consideration.The eDNA concentration may not always directly correspond to the actual abundance or biomass of fish, as various factors can influence the release, decay, and transport of eDNA in the environment; (b) Biotic factors.Intraspecific variation in DNA production among fish individuals can affect the accuracy of eDNA-based abundance estimation.Stress, metabolism, size, distribution, density, feeding habits, reproduction, and fish migration patterns can influence the amount of eDNA released into the environment; (c) Abiotic factors.Abiotic factors also play a role in eDNA dynamics.Water flow, temperature, and depth can affect the dispersal and degradation of eDNA in aquatic systems.Understanding the influence of these factors is crucial for accurate abundance estimation; (d) Environmental DNA decay.The decay rate of DNA in the environment can vary depending on water conditions, microbial activity, and other environmental factors.This decay process can impact the detectability and reliability of eDNA-based abundance estimation; (e) Spatial and temporal scales.The spatial and temporal scales at which eDNA sampling is conducted can influence the results.eDNA concentrations may vary across different locations and periods, requiring careful consideration of sampling strategies to capture a representative picture of fish abundance; (f) Methodological considerations.The methods for capturing, extracting, and amplifying eDNA can introduce biases and limitations.Standardization of protocols and optimization techniques are necessary to ensure accurate and comparable results.These challenges highlight the importance of considering various biotic and abiotic factors, as well as methodological considerations when using eDNA approaches for estimating fish abundance.Careful study design, appropriate primer design, and rigorous data analysis can help mitigate these limitations and improve the reliability of eDNA-based abundance estimation in fish monitoring and conservation efforts.

Conclusion
This review demonstrates the significant potential of eDNA technology in detecting and monitoring threatened fish species in Indonesian waters.The study reveals a high number of threatened fish species in Indonesia, with several families showing a particularly concerning level of threat.The successful use of eDNA technology in detecting threatened fish species highlights its effectiveness as a tool for species detection and distribution mapping.However, the review also emphasizes the need for further development and validation of eDNA techniques for accurately estimating fish abundance.The study identifies several obstacles and limitations in estimating fish abundance using eDNA approaches, including careful interpretation, biotic and abiotic factors, eDNA decay, spatial and temporal scales, and methodological considerations.Addressing these challenges through careful study design, standardization of protocols, and optimization of techniques will improve the reliability of eDNA-based abundance estimation in fish monitoring and conservation efforts.This comprehensive review calls for ongoing research and conservation efforts to harness the full potential of eDNA technology in protecting threatened fish species and preserving Indonesia's rich aquatic biodiversity.

Supplementary Table 2. Threatened Fish Species in
Larashati et al. (2021 unpublished) and Kusumah et al. (2023 unpublished) started the research on the development of eDNA techniques for detecting the population or abundance of threatened fish species.Larashati et al. focused on the Near Threatened (NT) eel species Anguilla bicolor, while Kusumah et al. worked on the threatened balashark fish (Balantiocheilos melanopterus).

Table 6 . 2 .
Species-specific eDNA development stages for abundance studies conducted by Larashati et al. and Kusumah et al. Specific primers & a probe design for target species In silico and in vitro tests √ √ 3. Laboratory/pond validation Experiment in the aquaria or water sampling in the pond √ … 4. Field validation Water sampling in the natural habitats … … 5. eDNA application for detecting and monitoring Application in the field … … A.b. = A. bicolor; B.m. = B. melanopterus

Table 1 .
Keywords utilized in this review.

Table 3 .
Indonesian threatened fish detected by eDNA.

Table 4 .
Primers are used to detect Indonesian threatened fish.

Table 5 .
Another threatened fish in the world has been detected using eDNA.