Received: 20 December 2022 Revised: 12 June 2023 Accepted: 4 July 2023

DOI: 10.1111/conl.12971

LETTER

Shark-dust: Application of high-throughput DNA

sequencing of processing residues for trade monitoring of
threatened sharks and rays
Andhika P. Prasetyo1,2,3 Joanna M. Murray4 Muh. Firdaus A. K. Kurniawan5
Naiara G. Sales1 Allan D. McDevitt1,6,† Stefano Mariani7,†
1 School of Science, Engineering and Environment, University of Salford, Salford, UK
2 Centre Fisheries Research, Ministry for Marine Affairs and Fisheries, Jakarta, Indonesia
3 Research Centre for Conservation of Marine and Inland Water Resources, National Research and Innovation Agency, Bogor, Indonesia
4 Centre for Environment, Fisheries and Aquaculture Science, Lowestoft, UK
5 Directorate for Conservation and Marine Biodiversity, Ministry for Marine Affairs and Fisheries, Jakarta, Indonesia
6 Marineand Freshwater Research Centre, Department of Natural Resources and the Environment, School of Science and Computing, Atlantic
Technological University, Galway, Ireland
7 School of Biological and Environmental Sciences, Liverpool John Moores University, Liverpool, UK

Correspondence
Andhika P. Prasetyo, School of Science, Abstract
Engineering, and Environment, Illegal fishing, unregulated bycatch, and market demand for certain products
University of Salford, Salford, UK.
Email: [email protected], (e.g., fins) are largely responsible for the rapid global decline of shark and ray
[email protected] populations. Controlling trade of endangered species remains difficult due to
Stefano Mariani, School of Biological and product variety, taxonomic ambiguity, and trade complexity. The genetic tools
Environmental Sciences, Liverpool John traditionally used to identify traded species typically target individual tissue
Moores University, Liverpool, UK.
Email: [email protected] samples, and are time-consuming and/or species-specific. Here, we performed
high-throughput sequencing of trace DNA fragments retrieved from dust and
Funding information
scraps left behind by trade activities. We metabarcoded “shark-dust” samples
Illegal Wildlife Trade (IWT) Challenge
Fund, Grant/Award Number: IWT057 from seven processing plants in the world’s biggest shark landing site (Java,
Indonesia), and identified 61 shark and ray taxa (representing half of all chon-
drichthyan orders), more than half of which could not be recovered from tissue
samples collected in parallel from the same sites. Importantly, over 80% of shark-
dust sequences were found to belong to CITES-listed species. We argue that
this approach is likely to become a powerful and cost-effective monitoring tool
wherever wildlife is traded.

KEYWORDS
DNA metabarcoding, elasmobranchs, environmental DNA, Indonesia, trade monitoring

† These authors contributed equally.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
© 2023 The Authors. Conservation Letters published by Wiley Periodicals LLC.

Conservation Letters. 2023;16:e12971. wileyonlinelibrary.com/journal/conl 1 of 10

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2 of 10 PRASETYO et al.

1 INTRODUCTION (Shivji et al., 2002) and mini-barcoding (Fields et al.,

2015) can robustly identify species in fresh and processed
Continued and increasing anthropogenic stressors have samples, while real-time qPCR (Cardeñosa et al., 2018),
devastated habitats and wildlife across the globe, includ- LAMP-based (But et al., 2020), and universal close-tube
ing the dramatic depletion of sharks and rays (hereafter barcoding (Prasetyo et al., 2023) assays can detect target
also referred to as “elasmobranchs”) (Dulvy et al., 2021). species in a matter of hours.
Conservative life histories (Mardhiah et al., 2019) make All these methods require the collection and analysis
elasmobranchs vulnerable to fisheries overexploitation, of individual specimens, which is a significant limitation
and their extirpation can destabilize functional diversity when large volumes of samples, across many locations,
and ecosystem structure (Dulvy et al., 2021). Although must be inspected in a limited timeframe (Prasetyo et al.,
some elasmobranch fisheries can be sustainably managed 2021). Recent advances in next-generation sequencing
(Simpfendorfer & Dulvy, 2017), market demand for high- have shaped the transformation of general DNA barcod-
value products, such as fins, liver oil, and gill plates, typ- ing (Hebert et al., 2003) into a technique that allows the
ically leads to overexploitation of elasmobranch resources simultaneous identification of multiple taxa from an inor-
(Dulvy et al., 2021), which is then further fueled by illegal dinate mixture, known as DNA metabarcoding (hereafter
and unreported catches. referred to as just “metabarcoding”) (Riaz et al., 2011).
This combination of market demand, overexploitation, Metabarcoding has been broadly applied to analyzing envi-
and lack of detail in catch and trade data (Cawthorn et al., ronmental DNA (eDNA) samples—trace DNA fragments
2018) requires effective mechanisms to monitor elasmo- left behind by organisms in water, soil, and air, an approach
branch populations and ensure their sustainable manage- that effectively complements, and in some cases surpasses,
ment (Prasetyo et al., 2021). This includes improved catch traditional monitoring (Aglieri et al., 2021; Boussarie et al.,
reporting, special regulations for endangered species— 2018). Such developments are unlocking novel applica-
such as the Convention on International Trade in Endan- tions in trade monitoring, allowing bulk mixtures to be
gered Species of Wild Fauna and Flora (CITES) (Pavitt analyzed, and tackling the limitations of existing tools.
et al., 2021)—and a range of other transdisciplinary initia- Here, we propose a novel metabarcoding application, by
tives (Booth et al., 2019). A critical step in this context is the targeting seven key shark and ray trading hubs in the island
accurate reconstruction of the biodiversity composition of of Java, Indonesia, the top elasmobranch-landing country
elasmobranch products at landing sites, processing plants, in the world (FAO, 2022). The seven sites were selected by
markets, and export hubs. considering their importance in trade flows (role and vol-
In 2023, the challenges facing trade biodiversity moni- ume, Prasetyo et al., 2021) as well as accessibility. We used
toring have more than tripled, as the number of CITES- high-throughput metabarcoding to screen the byproducts
listed species has increased from 47 to 151 (CITES, 2022a); of processing plant activities (which we term “shark-dust”)
yet, species listed in Appendix II can still be traded, by and compare them with single-specimen barcoding. This
considering the viability of exploitation within the Non- unconventional application is poised to minimize labor
Detriment Findings (NDF) framework (Smith et al., 2011). requirements, enhance the detection of species that are
Thus, conservation managers now face a scenario where not visible at the time of inspection, and be implemented
14% of the 1120 described elasmobranch species—nearly globally.
one-third of which deemed to be under some level of con-
servation threat (IUCN, 2021)—can still be traded through
the application of the NDF mechanism and substituted 2 MATERIALS AND METHODS
for other species (a form of species/product mislabeling).
Understanding and regulating trade in these species is 2.1 Sample collection
challenging because elasmobranch products are extremely
diverse in both their usage and their value, and are pro- Dust collections and shark/ray tissue samples (Figure 1)
cessed in a myriad of different ways (Dent & Clarke, 2015). were collected from January to February 2020. Here,
Due to their similarity in appearance and lack of distinctive we targeted seven locations across cities on Java Island,
features in most derivative products, shark and ray species Indonesia (Figure S1), the main export hub for various
can be deliberately or accidentally mislabeled by those commodities, including elasmobranch products. We col-
involved in the trade (Figure 1). This has incentivized the lected two sets of samples: first, using gloves, we gathered
use of genetic identification methods, which progressively 28 mixtures (number of samples per location varies due
made DNA-based inference a staple of wildlife foren- to accessibility) of residual material from floors where
sics (Domingues et al., 2021). Of these, DNA barcoding shark products were processed, sorted, and stored for later
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRASETYO et al. 3 of 10

F I G U R E 1 The diversity of elasmobranch products on sale can lead to misidentification and mislabeling. Our samples included, among
others: (a) shark-dust from a pile of small dried fins, (b) tissue sample from a finless juvenile scalloped hammerhead shark whose cephalofoil
(the distinctive “face” in this Family, also known as “blade”) had been cut, (c) trunk (headless-finless shark body), (d) ray’s wing, (e) piles of
dried shark skin, (f) diced meat from shark heads, (g) shredded fin, (h) shark cartilage, (i) mobulid gill rack, and (j) lower lobes of shark
caudal fins.

shipping, henceforth referred to as “dust” samples (Table 2.2 Laboratory and bioinformatic
S1); then, we selected 183 tissue samples from individual procedures
specimens (Table S2). Replicated samples (4 ± 3 samples)
were collected in seven locations representative of Indone- DNA was extracted from all samples (dust and tis-
sia’s processing, export, and regulatory activity. About 10 sue samples) following the Mu-DNA protocol for
grams (about two tablespoons) of dust were scooped and tissue samples (Sellers et al., 2018). All DNA extrac-
stored at room temperature in sterilized 5 mL Click-Seal tions were diluted to 10–15 ng/μL prior to DNA
flat bottom tubes without a preservative. From the same amplification. The Elas02 primer pair (Elas02-F,
location, about 2 × 1 × 0.5 cm of tissue was collected from 5′-GTTGGTHAATCTCGTGCCAGC-3′; Elas02-R, 5′-
individual specimens, trying to cover the broadest possi- CATAGTAGGGTATCTAATCCTAGTTTG-3′) was used to
ble spectrum of morphological diversity observed at the target an ∼180 bp amplicon from a variable region of the
sites, including both fresh and processed products. The tis- 12S rRNA mitochondrial gene (Miya et al., 2015; Taberlet
sue was then stored in 2.0 mL screw-cap microcentrifuge et al., 2018). Given that dust was sampled from the floor,
tubes, submerged in 90% ethanol, and stored at 4◦ C. The an elasmobranch-specific 12S marker was selected to avoid
sampling tools were either changed or sterilized each time nontarget amplification, as the use of a COI-based marker
between samples. would likely lead to the vast majority of reads coming
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4 of 10 PRASETYO et al.

from other organisms (Collins et al., 2019). Samples (S. lewini) could be found almost everywhere and was most
were amplified in triplicate to minimize amplification prevalent in the processing plants in Indramayu (IDM2
stochasticity, and replicates were later pooled into a single and IMD3), Banyuwangi (BYW7), and Surabaya (SBY6).
representative sample. Meanwhile, the sequencing of Spot-tail shark, recently added to the CITES list, showed
individual tissue samples followed a massively parallel the highest read abundance in the Indramayu processing
framework hereafter termed “high-throughput barcod- plants (Figure 2b). Among non-CITES-listed species, tiger
ing”. The Leray-XT primer pair targeting an ∼313 bp shark (Galeocerdo cuvier) was the predominant species
amplicon from a region of the COI mitochondrial gene across sampling locations, followed by zebra shark (Stegos-
(Wangensteen et al., 2018) was used for DNA amplification toma fasciatum), the Australian weasel shark (Hemi-
from tissue samples. galeus australiensis), whitespotted whipray (Himantura
Adapters were ligated to PCR products using the KAPA gerrardi), and spotless smooth-hound (Mustelus griseus)
HyperPrep Kit PCR-Free, and then quantified by qPCR (Figure 2c). These five species contributed about 70% of
using the NEBNext R Library Quant Kit for Illumina the non-CITES-listed read count overall, but their relative
sequencing. The dust-generated library was diluted to 9 proportions varied greatly among locations.
pM, and sequenced on an Illumina MiSeq run using a The prevalence and abundance of reads from CITES-
2×150 bp v2 kit; the tissue sample libraries were diluted to listed species detected in dust samples show that these
18 pM and sequenced on an Illumina MiSeq run using a animals continue to be major trade commodities and that
2×300 bp v3 kit. PhiX spike was at 1% for both runs. Bioin- monitoring efforts need to be intensified. Such species of
formatic analysis was based on OBITools, a python-based conservation concern—primarily pelagic taxa—are found
pipeline specifically designed for analyzing massively par- in abundance in processing plants (IDM2, IDM3, CLP4,
allel sequencing data in a DNA metabarcoding context and BYW7) and exporter warehouses in the main export
(Boyer et al., 2016), with taxonomic assignment conducted hub cities (i.e., Jakarta and Surabaya—JKT1 and SBY6).
against a custom reference database (Table S3). Details These results corroborate earlier indications that CITES-
on laboratory, bioinformatic, and statistical procedures listed species, such as thresher sharks, hammerhead
can be found in the Supplementary Materials, and the sharks, silky sharks, wedgefishes, and guitarfishes, are still
scripts and dataset associated with the study are pro- being traded in major Indonesian markets (Fahmi et al.,
vided in a dedicated GitHub repository (https://github. 2021) and may still be exported through NDF mechanisms
com/andhikaprima/sharkdust) which has been archived (CITES, 2022b). In Hong Kong, which is the main des-
(https://doi.org/10.5281/zenodo.7997300). tination market, fin products of CITES-listed species are
modeled to be ∼10% of the overall traded volume (Fields
et al., 2017). Based on our results from the world’s largest
3 RESULTS AND DISCUSSION exporter—and the recent expansion of CITES listings—
these figures are likely an underestimation. Dust samples
3.1 Dust metabarcoding analysis also contained the DNA of several key reef-associated
sharks, such as blacktip reef shark (C. melanopterus),
We obtained 5,580,616 reads from 28 discrete dust samples. whitetip reef shark (Triaenodon obesus), and sand tiger
We refined the final dataset (by removing contaminants shark (Carcharias taurus). These species play an impor-
and non-elasmobranch reads) to 4,640,239 (83.15% of ini- tant part in the equilibria of coral reef ecosystems, which
tial reads) elasmobranch-only reads, partitioned into 61 is particularly concerning for Indonesia, where reef sharks
molecular operational taxonomic units (MOTUs) (Figures have been driven to near functional extinction (MacNeil
S1, S2, S5, and Table S4) belonging to seven differ- et al., 2020). Several mesopredatory ray species were also
ent orders: requiem sharks (Carcharhiniformes), mack- detected, including Hortle’s whipray (Himantura hortlei),
erel sharks (Lamniformes), dogfish sharks (Squaliformes), mangrove whipray (Himantura granulata), pale-edged
cow sharks (Hexanchiformes), carpet sharks (Orectolobi- stingray (Dasyatis zugei), and bluespotted stingray (Neotry-
formes), stingrays (Myliobatiformes), and shark-like rays gon kuhlii). These species, albeit not controlled under
(Rhinopristiformes). Taxonomic assignment successfully CITES, significantly contribute to trophic interactions in
identified 54 of the 61 MOTUs to species level, with five key coastal ecosystems (Flowers et al., 2021); in fact, 90% of
assigned to genus level and two only attributable to family non-CITES-listed species detected from dust samples are
level. currently designated as threatened species under the IUCN
Nearly, 84% of the filtered reads belonged to 32 CITES- (International Union for Conservation of Nature) Red
listed taxa, including high-profile pelagic bycatch species, List (IUCN, 2021). Therefore, beyond trade enforcement
such as hammerhead sharks (Sphyrna spp.), silky shark aspects, obtaining information on these taxa is critical
(Carcharhinus falciformis), and spot-tail shark (Carcharhi- for monitoring the impact of exploitation on population
nus sorrah) (Figure 2a). The scalloped hammerhead shark dynamics and ecosystem health.
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRASETYO et al. 5 of 10

F I G U R E 2 The species composition of CITES and non-CITES-listed species (square-rooted and normalized read abundance) across
sampled locations (a), composition of CITES-listed species (b), and composition of non-CITES-listed species (c). The top 5 species are
visualized with silhouettes and the same color in the bar chart after data transformation and normalization.

3.2 Comparison of species detections taxa across methods; however, the dust samples detected 17
from dust and tissue samples more genera than tissue samples, and uniquely identified
10 CITES-listed species (Figures 3b and 4a, Table S5). When
Tissue-based barcoding successfully identified 175 out of sequencing reads from the dust samples were transformed
183 samples associated with the locations where dust sam- into presence and absence data, species compositions
ples were taken. Specimens were partitioned into 36 taxa, between dust and tissue samples were shown to be sig-
nearly all of which were also detected in the dust samples nificantly different (PERMANOVA: F = 3.49, p = 0.001;
(Figure 3a). Overall, we were able to identify more than 64 Figure 3c and Table S6). Tissue samples showed a greater
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 of 10 PRASETYO et al.

F I G U R E 3 Comparison between species recovery from dust and tissue samples; Venn diagrams of all elasmobranch species (a),
CITES-listed species only (b), and non-metric multidimensional scaling (nMDS) based on Jaccard similarity index between two sample types
in different locations (c). Samples have been pooled into the seven locations. Nb. Only species-level taxa are considered except for Mobula sp.
and Rhynchobatus sp. as these taxa, detected by dust metabarcoding, could only be confidently assigned to genus using the 12S marker. The
stress value measures how well the original distance matrix between samples is reduced in two dimensions. Values below 0.2 are generally
considered a good representation. Moreover, the size of the circles on the NMDS plot is proportional to species richness (number of species
being detected in a particular location and sample type).

separation among locations, due to the high-grading bias spp.). The latter three are hardly ever seen at landing
introduced by the single-specimen approach to sampling places, given their fully protected status under Indonesia’s
(which may also select for more “notable” samples). Dust regulations (Ministerial Decree No. 61 in 2018 concerning
samples showed a consistently greater species richness the utilization of protected and/or CITES-listed species,
across locations, detecting an average of 31.57 (± 16.34) Prasetyo et al., 2021). These findings mirror the perfor-
taxa per collection, with tissue samples averaging 11.14 mance of eDNA studies on elasmobranchs from natural
(± 6.01), as is also shown by the taxon accumulation curve environments, which consistently reveal important “dark
(Figure 4b). diversity” that is missed by pre-existing biomonitoring
Dust metabarcoding has much greater power to unveil tools (Boussarie et al., 2018). In this sense, the “shark-dust”
a comprehensive portrayal of shark and ray species metabarcoding approach can boost and streamline all the
being traded, for a considerably lower sampling effort biodiversity, fishery, and trade control operations that have
(Ndust = 28 vs. Ntissue = 175) and less disruption of the up to this point been carried out via earlier-generation
processing and trading operations in the visited hubs DNA monitoring tools.
(Figure 4c). Dust samples revealed some cryptic and rare There were 40 CITES-listed taxa identified in total, with
species, such as winghead shark (Eusphyra blochii), pigeye 22 taxa—including thresher sharks (Alopias spp.), mako
shark (C. amboinensis), sand tiger shark (Carcharias tau- sharks (Isurus spp.), and two hammerhead species—that
rus), smooth hammerhead (S. zygaena), knifetooth sawfish are commonly found at landing sites (S. lewini and S.
(Anoxypristis cuspidata), manta, and devil rays (Mobula mokkaran) identified using both dust and tissue samples.
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
PRASETYO et al. 7 of 10

F I G U R E 4 The cladogram (a) was generated using FigTree 1.4.4 using NADH2 region sequences (Naylor et al., 2012) from the NCBI
database. Colors represent sample type, such as dust samples (ORANGE) and tissue samples (BLUE) for results from each sampling location,
with CITES-listed species written in RED. Species accumulation curves (b) emphasize the differences in taxon detection rate between
methods; differences in sampling effort (c) are also visualized.

One of the tissue samples belonged to a species that is not two families and five genera with species listed in CITES
distributed in Indonesian waters: porbeagle shark (Lamna appendices, namely, wedgefishes (Rhynchobatus sp.), devil
nasus); but this was a single sample obtained from the rays (Mobula sp.), requiem sharks (Carcharhinus sp.), and
exporter’s reference collection that was used for education guitarfishes (Rhinobatinae). We had anticipated this issue
purposes. by developing an additional 12S reference database for our
analyses, but recent studies (Mariani et al., 2021; Miya
et al., 2020) had already shown that the size (170–180 bp)
3.3 A cutting-edge tool for trade and resolution of the 12S Elas02 fragment will not allow
monitoring discrimination between some closely related species, as
shown for Rhynchobatus, Mobula, Rhinobatinae, and also
Our findings showed that trade monitoring using dust for some species in the polyphyletic genus Carcharhinus
metabarcoding expands the reach of traditional barcoding (Sorenson et al., 2014). Yet, despite these limitations, the
methods. However, seven MOTUs could not be identified marker used remains the most effective metabarcoding
to species level from dust samples (Table S7), including tool for elasmobranch identification while also avoiding
 1755263x, 2023, 5, Downloaded from https://conbio.onlinelibrary.wiley.com/doi/10.1111/conl.12971 by Nat Prov Indonesia, Wiley Online Library on [29/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 10 PRASETYO et al.

nontarget amplification (Collins et al., 2019), and this list of 151 species (CITES, 2022a), which now include over
could be further strengthened through the ongoing expan- 50 species of requiem sharks (Carcharhinus spp.), over
sion of 12S and mitogenomic reference libraries (Collins 50 species between wedgefishes and guitarfishes, as well
et al., 2021) and the development of further taxon-specific as thresher sharks, hammerhead sharks, manta rays, and
assays, which may in the future accurately distinguish freshwater stingrays, the difficulties that countries face
between the most closely related species. in complying with CITES regulations have never been
Another advantage of bulk metabarcoding of process- greater. Decades of overexploitation have devastated elas-
ing byproducts includes the ability to detect trace DNA mobranch populations; but the use of trade bans will only
in situations where the original tissue source is no longer be successful in tandem with the implementation of reli-
available, either due to the complexity of trading opera- able and cost-effective monitoring tools. Indonesia, the
tions or as a result of deliberate concealment (Challender global epicenter of shark and ray trade, was the best place
et al., 2015). This may also allow for coarse estimation to assess the effectiveness of shark-dust metabarcoding
of relative volumes traded (Ershova et al., 2021; Shel- to improve trade monitoring. With this novel approach
ton et al., 2023), which would be impossible through the proving successful, the Ministry for Marine Affairs and
pain-staking tissue sampling from individual specimens. Fisheries immediately invested in certification schemes
Finally, dust metabarcoding is also more cost-effective: and biotechnological facilities, with the view of adopting
the collection of several dry processing residues is easier these DNA-based monitoring tools to improve sustain-
than collecting and preserving hundreds of individual tis- ability in the sector. The present approach based on the
sue samples. Along with the significant reduction in both residues of shark and ray processing activities should
time and costs of processing these dust samples in the lab effectively assist conservation strategies, working toward
right through to high-throughput sequencing, this much- the sustainability of elasmobranch populations across the
reduced sample size is then sufficient to garner higher world, and inspiring the design of similar methods to
species richness estimates than the individual-based tis- combat a wealth of other illegal wildlife trading activities.
sue analysis (Figure 4c). Technically, the collection of dust
residues, compared to tissue sampling, is open to envi- AC K N OW L E D G M E N T S
ronmental contamination, whereby DNA traces can be This study was funded by the Illegal Wildlife Trade (IWT)
detected from species that had passed through the sam- Challenge Fund number IWT057 through collaboration
pled establishment days, weeks, and potentially months between the University of Salford (UoS), the Ministry
earlier. Still, this “contamination” is an inherent feature for Marine Affairs and Fisheries (MMAF)—Indonesia,
of the approach, which purposely seeks to investigate the the Centre for Environment, Fisheries and Aquaculture
biodiversity extracted, processed, and traded through a Science (Cefas), the Rekam Nusantara Foundation—
given hub. Certainly, a formal framework will be required Indonesia, and Liverpool Jhon Mores University (LJMU).
and agreed by key stakeholders (traders, exporters, and We also thank the editor and two anonymous reviewers for
inspectors) on how to operationally implement shark-dust; providing constructive criticism.
one possible step would be to ask exporters to use brand-
new/cleaned containers for each batch of exports. Another D A T A AVA I L A B I L I T Y S T A T E M E N T
useful approach would be to identify robust, conservative The scripts and dataset associated with the study are pro-
“thresholds for detection” parameters in the bioinformatic vided in a dedicated GitHub repository (https://github.
workflow. com/andhikaprima/sharkdust) which has been archived
Recent developments in fast and portable technolo- (https://doi.org/10.5281/zenodo.7997300).
gies open up new opportunities to run metabarcoding
in the field. Our existing approach relies on laboratory ORCID
equipment, which may be prohibitive in some contexts, Andhika P. Prasetyo https://orcid.org/0000-0002-7412-
especially in developing countries. Optimization of third- 8432
generation sequencing technologies (Johri et al., 2019; van Stefano Mariani https://orcid.org/0000-0002-5329-0553
der Reis et al., 2023) will most likely advance in situ bulk
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