The study examined environmental DNA from an urban stream and compared metabarcoding with species-specific qPCR for Eurasian otter detection. Fieldwork and literature reviews documented 13 mammal species, of which eDNA metabarcoding detected six, including Canis lupus, Felis silvestris and Mustela sibirica. The failure of metabarcoding to register otters in both sampling months, despite their confirmed presence and successful amplification by qPCR, led the authors to identify qPCR as the more effective method for investigating otter distribution in the urban setting.

Metabarcoding results for fish were similar to those of traditional surveys. At the family level, Cyprinidae represented 54.57% of fish species in April and 43.58% in October, followed by Gobiidae at 16.90% and 22.88%, respectively. Pseudorasbora parva was the dominant fish species at the species level in Saetgang, accounting for 5.68% and 6.35% of relative abundance in April and October. The authors described this prey availability as potentially supporting opportunistic feeding by Eurasian otters, rather than presenting the metabarcoding results as direct evidence of otter diet.

The reported evidence is limited to one urban stream, two sampling months and the findings available in the abstract, so it does not establish year-round seasonal patterns or generalizability to other waterways. Within that scope, the study supports choosing qPCR for otter detection and metabarcoding for describing the surrounding fish assemblage. The authors concluded that the suitable eDNA analysis method depends on the research purpose.

Subject of Research: Comparison of metabarcoding and qPCR for seasonal Eurasian otter eDNA detection in an urban stream

Article Title: Optimising eDNA analysis for urban otter monitoring: seasonal patterns, detection strategies and prey availability

Article References: Kang, Y., Han, S., & Song, Y. (2024). Optimising eDNA analysis for urban otter monitoring: seasonal patterns, detection strategies and prey availability. Metabarcoding and Metagenomics, 8, Article e115512. https://doi.org/10.3897/mbmg.8.115512

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.115512

Keywords: environmental DNA, Eurasian otter, metabarcoding, qPCR, urban stream, seasonal monitoring, fish prey, Pseudorasbora parva, Optimising, eDNA, analysis, urban

The authors outline steps in the eDNA metabarcoding workflow that can influence taxonomic assignments and discuss the strengths and weaknesses associated with each stage. They also describe how protocols can be developed to support species identification and identify communication strategies for stakeholders and end users. The central emphasis is on aligning methodological decisions with the objectives of each project.

The review identifies several limitations that can compromise interpretation: incomplete reference databases, contamination risks, ambiguous sequences and detection biases. These constraints mean that assigning a species from metabarcoding sequences is not a wholly automatic process and may involve unresolved uncertainty. The supplied abstract does not quantify the prevalence or effect of these problems.

The authors argue that transparent reporting of methods and limitations, together with clear communication, is critical to the reliable use of eDNA and other metabarcoding or amplicon-sequencing results. They state that such transparency can support accuracy, reliability and usability in conservation and management decisions, while fostering trust among users. Because the available evidence is an abstract of a review and perspective, it does not establish a new consensus framework or demonstrate improved identification outcomes through a controlled evaluation.

Subject of Research: Review of species identification practices in environmental DNA metabarcoding

Article Title: Guidance and best practices for species identification using eDNA metabarcoding – When do you call a cod a cod?

Article References: Watts, A. W., Gold, Z., Patin, N. V., Adams, N. G., Baker, J. D., El Baidouri, F., Grey, E., Holmes, A. E., Jacobson, K., Jungbluth, S., Kajita, T., Kiledal, A., Lemay, M. A., Montes, E., Muller-Karger, F. E., Miller, J., Ogburn, M. B., Pitz, K. J., Silliman, K., … Thompson, L. R. (2026). Guidance and best practices for species identification using eDNA metabarcoding – When do you call a cod a cod?. Metabarcoding and Metagenomics, 10, Article e174450. https://doi.org/10.3897/mbmg.10.174450

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.174450

Keywords: environmental DNA, eDNA, metabarcoding, species identification, taxonomic assignment, reference databases, contamination, detection bias, conservation, bioinformatics, Guidance, best

The field experiment was conducted in Bazaruto Archipelago National Park, where the largest known dugong subpopulation in the Western Indian Ocean occurs. Dugongs are large, mostly herbivorous marine mammals whose populations have declined throughout their range, and visual or aerial surveys can be difficult where animals are scarce, solitary, cryptic or avoid human activity. A positive eDNA result therefore offers a non-invasive way to supplement field observations, although the signal may reflect an animal that was present nearby or recently passed through the sampled area rather than one present at the exact sampling point and time.

During the campaign, the research team followed 12 non-linear boat tracks at an average speed of about 1.5 knots through known areas of dugong occurrence. The sampling design compared surface water with near-bottom water, filtered volumes of 1.5, 30 and 60 litres, and approximately 25 millilitres of sediment. Two filter configurations were used: 0.45-micrometre polyethersulfone Waterra cartridges for the 30- and 60-litre samples and 0.22-micrometre Millipore Sterivex filters for the 1.5-litre samples. Near-bottom water was collected about one metre above the seabed with a pump mounted on a manta tow frame, while surface samples were collected from the boat or from composite samples taken at the beginning and end of each track.

The investigators developed a species-specific qPCR assay, named Ddug01, targeting a 115-base-pair segment of the mitochondrial 16S ribosomal RNA gene. In silico analysis indicated amplification specific to dugongs and the extinct Steller’s sea cow. Testing with serial dilutions of dugong tissue DNA produced a linear response from undiluted DNA through a 10^-4 dilution, with a slope of -4.819, an R² of 0.984 and an amplification efficiency of 61.3%. The lowest concentration detected in all four technical replicates was 0.944 picograms of DNA per reaction, which was assigned as both the limit of detection and limit of quantification. Amplification became inconsistent at the next tenfold dilution and was absent at lower concentrations.

Each environmental sample was amplified in eight technical replicates, and a beta-binomial mixed-effects model accounted for positive versus negative replicates while including sampling batch as a random effect. Laboratory processing was associated with markedly stronger qPCR detection than field processing, with a significant treatment effect of χ² = 15.06 and p < 0.001. The comparison concerned complete operational workflows rather than extraction location alone: field samples were processed with smaller amounts of preservation buffer, lower centrifugation capacity and, for Sterivex filters, a different extraction kit. Positive results from field processing showed that the method functioned, but the lower yield indicates a practical limitation of the field protocol.

Filtration volume and vertical position interacted significantly, with χ² = 3.875 and p = 0.049. At the low volume of 1.5 litres, near-bottom samples had about 5.8 times higher odds of producing a positive qPCR replicate than surface samples, a statistically significant difference with p = 0.013. At 60 litres, however, detection was similar between the two vertical positions. The corresponding pattern appeared when volume was compared at each depth: increasing the filtered volume from 1.5 to 60 litres significantly improved detection in surface water, with an odds ratio of 5.49 and p = 0.012, whereas it did not significantly change detection near the bottom, with p = 0.917.

The highest sample-level detection rate came from 60-litre near-bottom samples processed in the laboratory, with 9 of 11 samples positive. The researchers also detected dugong DNA in samples collected 1 hour 40 minutes and 2 hours after the last visual sightings, and at two sites where no animals had been directly observed. One event without a visual sighting produced no eDNA detection, while a known feeding site with visible feeding trails also yielded no positive signal. These outcomes illustrate the complementary nature and limitations of the two approaches: eDNA may reveal animals missed by visual surveys, but absence of a detection does not necessarily establish that dugongs were absent, particularly where tides dilute signals or DNA disperses quickly.

Sediment did not outperform the water-based methods in this pilot. None of the 10 sediment samples produced a positive result, whereas every water-sampling and extraction combination produced at least one detection. The comparison is incomplete because sediment was processed only in the field, where overall detection was lower, and the extraction kit may have performed differently with sediment than with water. The result therefore does not establish that sediment is inherently unsuitable for dugong eDNA analysis. It shows only that the tested sediment workflow offered no advantage under the conditions encountered, while sediment eDNA may integrate signals over longer periods than water samples and could provide information about past rather than recent presence.

High-volume filtration also carried practical benefits. The 0.45-micrometre Waterra cartridge filtered water at an average rate of 25 seconds per litre, allowing a 60-litre sample to be processed in less than 25 minutes at the surface and underwater without clogging. Placing the cartridge directly on the pre-pump side of the system also reduced the amount of single-use tubing consumed, a relevant advantage for large monitoring programs. The field protocol used centrifugation limited to 5,000 g and could not process the full preservation-buffer volume processed in the laboratory. Such conditions are less effective for recovering free or weakly adsorbed DNA than higher-force centrifugation or precipitation methods involving reagents that are difficult to transport because of their toxicity and volatility.

The authors therefore favor mobile surface sampling with high-volume filtration as a practical approach for large-scale dugong monitoring, while acknowledging that the proof-of-concept study included only 12 sampling events and a limited number of positive detections. Sampling volume was also partly confounded with filter configuration and, in some laboratory comparisons, extraction kit, so the results cannot be attributed exclusively to volume. The study supports further testing across broader spatial, temporal and hydrodynamic conditions, including tidal cycles, and does not directly quantify DNA persistence or transport. Even with those limitations, the work shows that carefully chosen water sampling and processing conditions can improve the recovery of rare marine-mammal eDNA and could complement conventional surveys in tropical coastal habitats.

Subject of Research: Optimization of environmental DNA sampling and extraction for detecting rare dugongs in tropical coastal waters

Article Title: Optimizing Marine eDNA Protocols for Rare Megafauna: A Case Study on Dugongs in the Western Indian Ocean

Article References: Bernagou, L., Serval, A., Beudard, F., Mathey, J., Mercky, Y., Le Bozec, C., Trotzuk, E., Paunde, K., Garcia, M., Bellemain, E., & Corse, E. (2026). Optimizing Marine eDNA Protocols for Rare Megafauna: A Case Study on Dugongs in the Western Indian Ocean. Ecology and Evolution, 16(10), Article e74500. https://doi.org/10.1002/ece3.74500

Image Credits: AI Generated

DOI: 10.1002/ece3.74500

Keywords: dugong eDNA, environmental DNA, marine monitoring, qPCR, Bazaruto Archipelago, water filtration, molecular detection, Mozambique, sea mammal conservation, Optimizing, Marine, eDNA

Environmental DNA works by capturing the genetic material organisms constantly shed into their surroundings through skin, mucus, faeces, gametes, and decaying tissue. Researchers can filter water or collect soil, amplify the recovered DNA with primers targeting specific gene regions, and sequence the result to identify which species were present, all without capturing, handling, or even seeing a single animal. The approach is faster, cheaper, less destructive, and often more sensitive than traditional surveys such as gillnetting, electrofishing, or pitfall trapping, and it can detect rare and elusive species that conventional methods routinely miss.

To understand how this technology has been applied across the region, a team of Tanzanian researchers led by Joshua James Ibrahim of the Nelson Mandela African Institution of Science and Technology conducted a systematic review following PRISMA 2020 guidelines. They searched Scopus and Google Scholar for peer-reviewed English-language studies published between January 2018 and April 2026 that used eDNA or DNA metabarcoding to assess aquatic or terrestrial biodiversity in sub-Saharan African countries. Only twenty-five studies met the criteria, a remarkably small evidence base for a region of more than forty countries containing roughly thirty-four global biodiversity hotspots.

The geographic and taxonomic patterns in those studies are stark. Southern Africa dominated with twelve studies, or forty-eight percent of the total, driven largely by South Africa’s six studies. East Africa contributed seven, West Africa five, and Central Africa just two, both from the Democratic Republic of Congo and focused on pathogens. Notably, the Congo Basin, one of the planet’s most important rainforests, has no published eDNA biodiversity studies at all as of April 2026. Aquatic ecosystems accounted for eighty-eight percent of the research, fish were the target taxon in forty-eight percent of studies, and not a single study was dedicated to arthropods, the insects, spiders, and other invertebrates that make up the majority of the region’s undescribed animal species.

Methodologically, the field has settled into a narrow groove. Twelve percent of studies used the mitochondrial 12S rRNA marker, typically with fish-tuned primers such as MiFish or Teleo, and seventy-two percent relied on Illumina sequencing platforms. Water was the sampling medium in ninety-two percent of studies, with median field replication of three samples per site. Only sixteen percent of studies included seasonal replication, a serious omission for organisms whose communities shift dramatically across wet and dry seasons. This technical conformity around fish-targeted vertebrate markers has, in practice, narrowed the taxonomic reach of the region’s entire eDNA research program.

The single most cited limitation, reported in sixty-eight percent of studies, was incomplete reference databases, the curated libraries of DNA barcodes needed to match environmental sequences to named species. The consequences are quantifiable. In Tanzania’s Rufiji catchment, researchers found that twenty-nine of the ninety-one fish species estimated to occur there had no 12S reference sequence at all, meaning they were undetectable by eDNA regardless of how carefully the water was sampled. In South Africa, twenty-three percent of freshwater fish species lack COI barcodes and forty-one percent lack 12S barcodes. For arthropods the situation is worse: barcode coverage in public databases falls below five percent for many agriculturally critical groups such as parasitoid wasps, wild bees, and dung beetles.

Where reference libraries exist, the technology performs impressively. Studies detected sixty-six fish species in the Rufiji River, recovered twenty-seven elasmobranch species including twelve new records in Mauritania’s Banc d’Arguin National Park, and identified the invasive Nile perch in Nigerian waters. One Tanzanian study found that a single water sample could capture roughly forty percent of the species observed across fifty samples, suggesting that modest, well-replicated sampling can recover much of a detectable community. Comparisons with traditional methods show that eDNA and conventional surveys each detect species the other misses, so researchers increasingly view eDNA as a complement to, rather than a replacement for, nets and traps.

Logistics and capacity pose their own challenges. Field teams contended with security concerns, tidal mangrove channels, cold-chain failures, and filtration clogging, with one team averaging only 202 millilitres filtered per sample. Contamination risks surfaced in thirty-two percent of studies, in some cases requiring species to be excluded when they dominated extraction blanks. Perhaps most tellingly, seventy-six percent of the reviewed studies included non-African co-authors, and only twenty percent were led by Africa-based first authors, with several studies possible only through international funding. This capacity gap feeds a circular dependency: without local taxonomists, African reference barcodes cannot be produced; without barcodes, eDNA cannot assign species; and without detection success, funding for taxonomy remains scarce.

The review’s authors argue the way forward is clear. Reference database development should be prioritised for overlooked taxa, particularly agricultural pests and pollinators, with proposed initiatives modelled on existing global barcoding efforts. Emerging low-cost, portable sequencers such as Oxford Nanopore’s MinION, demonstrated in Nigeria for roughly one thousand dollars in capital cost compared with hundreds of thousands for Illumina systems, could democratise sequencing capacity and enable same-day pest detection in farm settings. Standardized multi-marker protocols, rigorous replication and controls, mandatory open-data deposit, and sustained Africa-led training and infrastructure investment round out the recommendations.

The findings arrive at a critical moment. Sub-Saharan Africa has lost about half its wetlands since 1900, and freshwater vertebrate populations declined by eighty-three percent between 1970 and 2016, the steepest drop of any continent. With fewer than five percent of African insect species formally described, the region cannot afford to leave entire ecosystems and taxonomic groups outside the reach of modern monitoring. The twenty-five studies synthesized here prove that eDNA works across African rivers, lakes, estuaries, and soils; the open question, the authors conclude, is whether funders and governments will invest in closing the reference gap and shifting the technology from its aquatic stronghold to the terrestrial and arthropod diversity on which the region’s food security depends.

Subject of Research: Applications and limitations of environmental DNA metabarcoding for biodiversity monitoring in sub-Saharan Africa

Article Title: Environmental DNA (eDNA) applications for biodiversity monitoring across countries in the Sub-Saharan African region: a systematic review

Article References: Ibrahim, J. J., Baino, A., Ndakidemi, B., Mbega, E., & Mkindi, A. (2026). Environmental DNA (eDNA) applications for biodiversity monitoring across countries in the Sub-Saharan African region: a systematic review. Frontiers in Conservation Science, 7, Article 1941471. https://doi.org/10.3389/fcosc.2026.1941471

Image Credits: AI Generated

DOI: 10.3389/fcosc.2026.1941471

Keywords: environmental DNA, eDNA, DNA metabarcoding, biodiversity monitoring, Sub-Saharan Africa, reference databases, conservation, freshwater fish, arthropods, terrestrial biodiversity, capacity building, Illumina sequencing

The research team, led by investigators mining public sequence data from NCBI GenBank, analysed sequences spanning ten orders of ray-finned fishes (Actinopterygii), including many commercially and ecologically important species. Their approach rested on two complementary datasets. The first drew on mitochondrial genomes, allowing the extraction of all nine target regions — the full 12S, 16S and COI genes plus the MiFish-U, Teleo, Berry-Fish, Ac16S, Folmer and Leray-Lobo amplicons — from the same individual organisms. This design eliminated a major confound: differences in reference sequence quality between markers could not distort the comparison, because every fragment came from identical source DNA. The second dataset used independently deposited sequences for each region, totalling thousands of species, to reflect what practitioners actually encounter when querying environmental reads against real-world reference libraries.

To quantify discriminatory power, the researchers constructed Neighbour-Joining trees for each fragment and measured the percentage of species that emerged as monophyletic — meaning all sequences of a species clustered together in a single clade. Monophyly is a stringent and intuitive criterion: if a marker cannot keep a species’ sequences together, downstream taxonomic assignment of environmental reads risks false positives and false negatives. The mitochondrial genome dataset comprised 2,094 sequences across 771 species, while the independent-sequence analysis expanded to 3,879 species, with some regions, such as Leray-Lobo, represented by nearly 50,000 locally aligned sequences.

The results from the mitochondrial genome analysis were striking. Among the metabarcoding amplicons, the COI Folmer and Leray-Lobo regions achieved the highest discriminatory power, with 89.2% and 87.0% of species monophyletic, respectively. At the opposite end, the 12S Teleo region — a fragment averaging under 70 base pairs — managed only 71.6%, the lowest of any amplicon tested. Full-length genes generally outperformed their shorter derivatives, as expected, since longer fragments harbour more informative sites. Yet the pattern was not simply a matter of length: the full 12S gene, roughly 950 base pairs, was outperformed by the shorter Folmer region (~658 bp) in every fish group, and even by the 313-base-pair Leray-Lobo fragment in three of five groups. The evolutionary rate of the region, not just its span, clearly matters.

That conclusion was reinforced by the congeneric divergence analysis. COI regions showed the highest average pairwise distances between species of the same genus, consistent with its status as a fast-evolving protein-coding gene. The authors attribute part of COI’s advantage to the accumulation of synonymous substitutions, which purifying selection tolerates more readily than amino-acid-changing mutations. Intriguingly, the Teleo region also displayed high congeneric divergence — it is a highly variable stretch of DNA — but its brevity prevents it from accumulating enough informative sites to build a strong phylogenetic signal. High variability in a very short fragment, it turns out, is no substitute for adequate length.

When the researchers turned to the independent-sequence datasets, the rankings inverted dramatically. Now the Ac16S amplicon led with 83.0% monophyletic species, while the Folmer and Leray-Lobo regions fell to the bottom, at 64.8% and 63.5%. The explanation lies not in the markers themselves but in the composition of public databases. COI’s enormous sequence volume — tens of thousands of records and the highest number of sequences per species — means it inevitably accumulates more misidentified or mislabelled entries. A single outlier sequence, likely stemming from a taxonomic error, can break a species’ monophyly. The authors documented cases such as the grouper Epinephelus areolatus, whose 79 sequences would have been perfectly monophyletic but for one likely misidentification. Because GenBank is frequently the direct source of reference data in eDNA workflows, these database-quality effects translate directly into real-world identification errors.

The study also probed why species fail to discriminate, categorising non-monophyly into species sharing identical haplotypes, sequences intruding into another species’ clade, outliers clustering away from their own clade, or combinations thereof. Short fragments such as Berry, Teleo and MiFish-U showed the highest proportions of non-monophyletic species sharing haplotypes with other species — a signature of genuinely insufficient resolution. In contrast, the COI regions showed elevated shares of species with both intruders and outliers, pointing more towards data-quality problems than intrinsic biological limits. This distinction matters: reference library gaps and errors can be fixed through curation, but a fragment that simply lacks informative sites cannot be improved by more data.

Biology itself imposes hard limits in some cases. The researchers found species that no mitochondrial marker could resolve, including the shads Alosa alosa and Alosa fallax, which hybridise extensively, and several tuna species in the genus Thunnus, where introgression and taxonomic ambiguity blur species boundaries. Scomber japonicus and Scomber colias were likewise indistinguishable across all nine regions, possibly compounded by outdated labels in sequence databases that predate the clarification of their geographic distributions. These cases underscore that mitochondrial DNA alone cannot untangle recently diverged or interbreeding species, regardless of the fragment chosen.

The practical implications are significant for anyone designing an eDNA survey. The authors argue that amplicon choice should weigh species discriminatory power alongside technical considerations such as primer specificity, amplification efficiency and fragment preservation in environmental samples. COI, despite its superior resolution, suffers from co-amplification of prokaryotes and non-target eukaryotes, reducing the fraction of usable reads; 12S and 16S amplicons like MiFish-U and Berry offer better fish specificity and shorter fragments that survive better in degraded environmental DNA. For species-poor systems where the fauna is known in advance, a single well-chosen amplicon may suffice. But in species-rich waters teeming with close congeners — precisely the conditions where fisheries stock assessment demands precision — the study advocates a multi-marker approach as standard practice, using one fragment’s results to rectify ambiguities in another’s.

Ultimately, the research delivers a sober but constructive message: eDNA metabarcoding’s effectiveness should not be overestimated when it relies on a single marker, especially under high diversity. Gaps in reference coverage will narrow as sequencing efforts continue, but differences in intrinsic discriminatory power are rooted in biology and will persist. By benchmarking nine genomic regions across hundreds of species and ten fish orders, this study gives the field the evidence base it needs to move from ad hoc marker choices towards consensus guidelines — a crucial step if environmental DNA is to fulfil its potential as a backbone of marine biodiversity monitoring and fisheries management.

Subject of Research: Species discrimination power of mitochondrial DNA markers and amplicons in marine fish eDNA metabarcoding

Article Title: Benchmarking the discrimination power of commonly used markers and amplicons in marine fish (e)DNA (meta)barcoding

Article References: Fontes, J. T., Katoh, K., Pires, R., Soares, P., & Costa, F. O. (2024). Benchmarking the discrimination power of commonly used markers and amplicons in marine fish (e)DNA (meta)barcoding. Metabarcoding and Metagenomics, 8, Article e128646. https://doi.org/10.3897/mbmg.8.128646

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.128646

Keywords: eDNA, metabarcoding, marine fish, COI, 12S rRNA, 16S rRNA, species discrimination, monophyly, reference databases, mitochondrial genomes, fisheries management, amplicon selection

An international team working under the eWHALE project, a collaborative initiative spanning the North-East Atlantic and the Mediterranean Sea, set out to answer a deceptively simple question: if four laboratories extract DNA from the very same water samples, do they get the same results? The answer, revealed through a rigorous inter-laboratory comparison known as a ring test, was not always. Despite similar total DNA concentrations across all participating facilities, one laboratory’s extraction protocol performed significantly worse at detecting the target species, and the discrepancy was only caught because the partners had agreed to test themselves against each other before the real monitoring work began.

The ring test involved four institutions: the University of Innsbruck in Austria, INRAE in France, University College Cork in Ireland, and the Institute of Marine Research in Norway. In summer 2023, each partner contributed water samples collected near marine megafauna in their home waters. Innsbruck supplied samples gathered near sperm whales in the Mediterranean and around the Azores, INRAE contributed samples taken near porbeagle sharks off Brittany, Cork collected water in the Shannon Estuary where bottlenose dolphins surface in groups, and the Norwegian institute sampled near basking sharks by the Lofoten Islands. In total, 39 eDNA samples entered the comparison.

The experimental design was elegant in its simplicity. Each laboratory first lysed its own filters, breaking open cells and releasing DNA into a storage buffer. Then, 250-microliter aliquots of every lysate were shipped to all four partners, where each facility extracted DNA using its own preferred protocol. The extracts were shipped back to the laboratory of origin for analysis. This exchange meant that every original sample could be measured four times over, once per extraction method, allowing researchers to isolate the effect of the extraction chemistry itself from the noise of field sampling.

The extraction methods themselves reflected the everyday reality of eDNA research. Innsbruck used a Qiagen BioSprint 96 Workstation, an automated platform that relies on paramagnetic beads to capture DNA. INRAE employed a Macherey-Nagel NucleoSpin Tissue Kit with repeated heated elutions, while Cork and the Norwegian institute both used the widely recommended Qiagen DNeasy Blood and Tissue Kit, albeit with different modifications, including vacuum-assisted filtration in Norway and an extended 56-degree incubation of lysates in Ireland. Each of these small choices, the study found, could ripple forward into the final检测结果, though the differences were often invisible when measuring total DNA alone.

To evaluate detection performance, the team developed three novel species-specific quantitative PCR assays, one each for sperm whale, porbeagle shark, and basking shark, using mitochondrial genes and a software pipeline called assayID that ranks candidate primer and probe designs by specificity and sensitivity. These TaqMan assays, alongside an existing SYBR Green assay for dolphin DNA developed at Cork, were validated in silico, in vitro, and finally against field samples, reaching Level 3 of the widely accepted five-level validation framework proposed by Thalinger and colleagues. The assays proved remarkably sensitive, with limits of detection as low as 0.00001 nanograms per microliter for dolphins, but sensitivity alone could not rescue poor extracts.

The statistical verdict was clear. Across 468 qPCR reactions, 115 amplified their target species. Total DNA concentrations measured by Qubit fluorometer did not differ significantly between laboratories, yet detection probability did. Extracts produced by the Norwegian laboratory amplified target species DNA significantly less often than those from the other three partners, in both an all-inclusive dataset and a conservative dataset restricted to amplifications at or above each assay’s limit of detection. When the team examined the protocols side by side, two subtle differences emerged between the Norwegian and Irish workflows, which otherwise used nearly identical kits: the extra hour of lysate incubation in Ireland may have improved DNA binding to silica membranes, while the vacuum system used in Norway may have compromised binding efficiency or left residual ethanol in the extracts.

Species identity mattered as much as laboratory identity. Sperm whale DNA was detected in 43 reactions and dolphin DNA in 56, while porbeagle shark yielded only 13 detections and basking shark a mere three, all vanishing entirely from the conservative dataset. The researchers attribute this pattern partly to biology: marine mammals slough skin and defecate at the surface between feeding dives, seeding the water with DNA, whereas sharks shed far less genetic material. Sampling design compounded the problem, as sperm whale samples were drawn from 10-liter volumes while shark samples came from only 5 liters, and some basking shark samples were likely collected too far from the animals themselves. A significant interaction between laboratory and species further suggested that extraction efficiency depends on the environmental character of each sample, meaning no single protocol is universally superior.

The study also offers a cautionary lesson about the limits of detection. Nearly 80 percent of the positive amplifications fell below the assays’ formal limits of detection, and excluding those low-level signals reduced overall detection rates by roughly 20 percent. Interpreting such borderline amplifications requires care, the authors argue, because treating them as false positives risks discarding real detections of genuinely rare species, while trusting them uncritically invites statistical noise. For cross-laboratory comparisons in particular, the conservative approach provides the fairest benchmark, since DNA extracted from the same environmental sample should theoretically be identical regardless of who processed it.

The practical outcome of the ring test was immediate: the Norwegian laboratory modified its extraction protocol for the remainder of the project, and the eWHALE partners now possess a documented, quantified understanding of how their methods compare. Three newly published qPCR assays are available for other eDNA practitioners targeting sperm whale, porbeagle shark, and basking shark. More broadly, the study stands as a template for what multinational eDNA monitoring should look like from the start, with validation exercises built in before any ecological conclusions are drawn. As eDNA moves from an academic curiosity toward routine conservation decision-making, the authors argue, this kind of inter-laboratory rigor is not optional overhead but the very foundation upon which comparable, trustworthy biodiversity data must rest.

Subject of Research: Inter-laboratory comparison of environmental DNA extraction protocols for detecting marine megafauna using novel species-specific qPCR assays

Article Title: Inter-laboratory ring test for environmental DNA extraction protocols: implications for marine megafauna detection using three novel qPCR assays

Article References: Rodriguez, L. K., De Bonis, L., McKee, J., McKenna, J. A., Urvois, T., Barbaccia, E., Dillane, E., Lanfredi, C., Hjellnes, H., Jung, A., Villa, E., Azzellino, A., Westgaard, J.-I., Quéméré, E., & Thalinger, B. (2025). Inter-laboratory ring test for environmental DNA extraction protocols: implications for marine megafauna detection using three novel qPCR assays. Metabarcoding and Metagenomics, 9, Article e128235. https://doi.org/10.3897/mbmg.9.128235

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.128235

Keywords: environmental DNA, eDNA, ring test, marine megafauna, qPCR, sperm whale, porbeagle shark, basking shark, dolphins, DNA extraction, laboratory standardization, eWHALE project

The research team, working with samples from Polish lakes and from the Mediterranean water column, compared the two sequencing strategies head to head. On the short-read side, they used Illumina MiSeq to sequence the V4 region of the 18S ribosomal RNA gene, the roughly 380-base-pair fragment that has become the de facto standard for protist metabarcoding. On the long-read side, they turned to Oxford Nanopore’s MinION platform, sequencing an amplicon of about 3,200 base pairs that spans the entire 18S gene, the internal transcribed spacers, the 5.8S gene and the beginning of the 28S gene. The comparison included a defined mock community of seven cultured protist species and complex environmental communities from ten freshwater and twelve marine samples.

Nanopore sequencing has long been viewed with caution in biodiversity research because of its higher error rate, which produces a substantial number of insertions and deletions that complicate the construction of clean taxonomic units. Denoising pipelines such as DADA2, which reliably separate biological signal from sequencing error in Illumina data, cannot model Nanopore’s less predictable error profile. The team’s answer is BaNaNA — the Barcoding Nanopore Neat Annotator — a Snakemake-based pipeline that clusters Nanopore reads into Operational Taxonomic Units rather than trying to denoise them. The pipeline basecalls reads with Dorado’s duplex super-accurate model, filters them for length and quality, extracts rDNA fragments with Barrnap, verifies the correct 18S-5.8S-28S structure with a custom Python script, and adjusts the clustering threshold to the average error rate of each sample. Consensus sequences are built with MAFFT alignments within clusters, polished with Minimap2 and Racon, screened for chimeras against the PR2 reference database and de novo, and finally clustered at 99 percent identity to produce the finished OTUs.

The mock community provided the clearest test of what each technology gets right and wrong. Illumina sequencing of the V4 region produced 268 ASVs with an average length of 379 base pairs, while Nanopore sequencing, processed through BaNaNA, yielded 147 OTUs averaging 1,843 base pairs. The Illumina ASVs gave a taxonomic composition that diverged substantially from the known community structure: dinoflagellates appeared most abundant even though they were added at low concentration, while Euglena gracilis, which made up nearly a third of the cells by count, was underestimated by three orders of magnitude. The full-length Nanopore OTUs tracked the expected composition far more closely, with haptophytes and euglenozoans appearing at roughly the right proportions. Crucially, the Illumina data also contained the highest number of false positives — sequences assigned to genera that were never part of the mock community — suggesting that the short-read denoising approach inflates apparent diversity with background noise.

Species-level identification told a similar story. Neither technology detected all seven mock species perfectly, but the full-length Nanopore OTUs achieved the highest number of species-level classifications. The advantage of sequence length became especially evident when the researchers extracted the V4 region from their Nanopore OTUs — creating so-called V4-tags — and compared species assignments. For Paramecium bursaria, the full-length OTUs placed the sequence correctly at species level, while many V4-tags could only be assigned to the genus level, causing the species’ apparent abundance to drop dramatically. The lesson is that longer fragments carry enough information for accurate species-level annotation, provided the reference database is adequate, while shorter fragments lose that resolution even when amplified with the same primers.

Environmental samples from the Masurian lakes and the Gulf of Lion confirmed the pattern in complex natural communities. Illumina sequencing recovered 1,668 ASVs from freshwater and 2,716 from marine samples, while BaNaNA processing of Nanopore data produced 1,068 freshwater and 1,230 marine OTUs averaging nearly 1,800 base pairs. At higher taxonomic levels, both technologies painted very similar pictures of community composition. In the lakes, dinoflagellates and perkinseans dominated, followed by ciliates, cryptophytes and chlorophytes, with little difference between sunlit and dark zones. In the marine samples, dinoflagellates and radiolarians prevailed, and the parasitic diplonemids were detected exclusively by Nanopore sequencing. Bray-Curtis beta diversity analysis grouped samples from both platforms into three distinct clusters corresponding to freshwater, marine photic and marine deep environments, indicating that the ecological patterns were robust regardless of which technology generated the data.

One of the study’s most striking findings concerns the sheer amount of undocumented protist diversity revealed by the long reads. The researchers identified 747 full-length 18S rDNA OTUs whose sequence identity to their closest PR2 reference fell between 80 and 97 percent — a range suggesting they represent taxa not yet in the database. Freshwater environments yielded 513 of these novel OTUs, representing 48 percent of all freshwater OTUs obtained, while marine samples contributed 234, or 19 percent of the marine total. Together, the novel sequences amount to 7.6 percent of all eukaryotic sequences currently in PR2 for freshwater and 3.5 percent for marine environments. Because the OTUs are full-length, they can be aligned and used to reconstruct phylogenetic trees. The resulting tree, built from 721 novel OTUs with IQ-TREE, grouped the sequences into clades spanning six supergroups and fifteen divisions, with most novel taxa belonging to perkinseans and dinoflagellates. The authors note that some of these sequences may represent entirely new clades, something impossible to detect with short amplicons that lack sufficient phylogenetic signal.

The study also highlights a persistent imbalance in protist reference databases. Freshwater samples showed roughly twice the proportion of low-identity sequences compared with marine samples — 41 percent versus 14 percent in the Nanopore data — a discrepancy the authors attribute directly to the underrepresentation of freshwater protists in PR2. Marine ecosystems have historically received more molecular attention, and the gap is visible at every taxonomic level. The researchers argue that filling this gap is essential for the future of long-read metabarcoding, since accurate annotation of full-length sequences requires databases with longer reference sequences. They also point out that Nanopore’s ability to sequence the entire rDNA operon will demand operon-spanning databases that are only now beginning to emerge.

Perhaps the most practically important result is the demonstration that short- and long-read data can be integrated. Because Nanopore OTUs cover the whole 18S gene, researchers can extract any primer-defined fragment — such as the V4 region — in silico and compare it directly with Illumina datasets generated years earlier with different primers. The V4-tags extracted from Nanopore OTUs proved highly comparable to Illumina V4 ASVs, opening the door to long-term biodiversity studies that combine vast archives of legacy Illumina data with new Nanopore surveys. The authors recommend retaining the taxonomic assignment from the full-length 18S rDNA before extracting any specific region, to fully exploit the resolution that long reads provide.

Taken together, the findings position Nanopore metabarcoding not merely as a cheaper complement to Illumina but as a reliable replacement for protist diversity studies. The full-length OTUs offer greater taxonomic resolution, richer phylogenetic information, fewer false positives and direct compatibility with existing short-read datasets. With BaNaNA freely available on GitHub and benchmarked on both mock and environmental communities, the study removes a key bioinformatic barrier that has kept long-read approaches on the margins of protist ecology. As Nanopore chemistry continues to improve — accuracy has risen from 97.6 percent with R9.4.1 flow cells to 99.1 percent with R10.4 — and as reference databases grow to match, the era of reading protist genes in full may finally be at hand.

Subject of Research: A new pipeline called BaNaNA shows that Nanopore long-read sequencing can match Illumina metabarcoding for protist diversity surveys while offering better species resolution, fewer false positives and phylogenetic power that short reads cannot deliver.

Article Title: From short to long reads: enhanced protist diversity profiling via Nanopore metabarcoding

Article References: Chwalińska, M., Karlicki, M., Romac, S., Not, F., & Karnkowska, A. (2025). From short to long reads: enhanced protist diversity profiling via Nanopore metabarcoding. Metabarcoding and Metagenomics, 9, Article e163750. https://doi.org/10.3897/mbmg.9.163750

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.163750

Keywords: protists, Nanopore sequencing, metabarcoding, 18S rDNA, biodiversity, BaNaNA, long-read sequencing, microbial ecology, eDNA, reference databases, short, long

Honeybee hives have long been recognised as rich sources of environmental nucleic acids, or eNA, encompassing both DNA and RNA from a wide range of taxonomic origins. Bees forage across landscapes, collecting pollen and nectar from flowering plants while simultaneously picking up microbial spores, fungal fragments, and viral particles. Genetic material from these sources accumulates within the hive, where it can be extracted and analysed to reveal the surrounding vegetation, detect plant pathogens, and assess honeybee health through the identification of pests and pathogens. Previous studies have used hive samples to monitor plant biodiversity, identify weed incursions, detect honeydew-producing insects, and even determine the regional provenance of honeys being sold commercially. However, the collection of internal hive samples such as honey, pollen, and bees requires specialists to open and handle hives, is seasonally dependent, and can weaken colonies over time if long-term monitoring is undertaken. Opening hives during colder months to collect samples can also risk colony survival.

The research team, working at sites in the Australian Capital Territory, set out to compare the effectiveness of non-invasive hive entrance swabbing with seven commonly used sample types. At a former pastoral property surrounded by grassland nature reserves and residential areas, they collected hive entrance swabs, hive debris swabs, honey, internal pollen, pollen trap samples, return forager bees, and top box bees across three sampling days in January 2023. Each hive entrance was swabbed using a sterile nylon FLOQSwab dipped in deionised water, rubbed across the entire length of the entrance four times while twisting to reach a range of depths inside the hive. Three consecutive swabs were collected per hive, along with the more invasive samples. All samples were immediately placed on ice in the field and stored at minus 80 degrees Celsius until DNA and RNA extraction, with no storage buffers used.

For DNA metabarcoding, the researchers amplified four genetic markers targeting bacterial 16S, animal COI, fungal ITS1, and plant ITS2 regions. The animal COI reactions included a peptide nucleic acid clamp specifically developed for this study to reduce amplification of honeybee DNA, allowing non-honeybee animal taxa to be detected more effectively. After quality control and filtering, over 5.2 million metabarcoding reads were obtained across the four markers. The results were striking: hive entrance swabs and hive debris swabs consistently recovered the highest number of taxa for bacteria, animals, and fungi. For the fungal ITS1 marker, hive entrance swabs detected 232 of the 309 total fungal taxa observed in the dataset, along with the highest total reads and average reads per sample. For bacteria, both swab types recovered 33 unique taxa each out of 35 total observed, while pollen trap samples produced the lowest number of total reads and unique taxa.

Plant detections told a slightly different story. Honey samples produced the highest average number of unique plant taxa per sample, closely followed by swab samples, with no significant differences observed between these sample types. Internal pollen samples, despite returning the highest total reads for the plant marker, yielded the lowest average number of unique taxa per sample. Return forager bees, which might be expected to carry the freshest plant genetic material, identified only 33 of the 62 total plant taxa detected. The researchers suggest this unexpected pattern may reflect the dynamic foraging behaviour of bees, which varies with the colony’s nutritional needs. Pollen is typically consumed more quickly than honey stores, meaning honey likely provides a cumulative record of the local environment while pollen samples give a more contemporary view of recent flowering activity.

For RNA virus detection, the team used metatranscriptomic sequencing on a subset of sample types collected in April 2024 from a separate apiary bordering Black Mountain and the Australian National Botanic Gardens. After quality control, 67 viruses were identified from a combined total of 17 million reads assigned to contigs. Honey samples had the highest observed number of viruses across all sample types, with an average of 23 viruses per replicate, followed by hive entrance swabs with 16 and top box bee samples with 14. Internal pollen samples contained the lowest observed number of viruses with an average of 7.73. All four sample types detected fungal and plant viruses, though honey samples comprised the highest number of plant viruses with 23 detected, followed by hive entrance swabs and top box bees with 10 each. Fungal viruses were most abundant in swab samples, with 10 detected. Honeybee-specific viruses were most abundant in bee samples, followed by honey and hive entrance swabs.

Five of the top 10 viruses detected across all sample types were associated with the plant genus Camellia, all detected in each sample type. The remaining five included two fungi-associated viruses, two viruses associated with clovers, and the honeybee virus black queen cell virus. The researchers note that the lack of honeybee and other insect viruses detected in pollen samples was surprising, given multiple other studies report success in detecting honeybee viruses using pollen. They attribute this result to the small number of hives sampled and a general low prevalence of honeybee viruses in these hives at the time of sampling. The trade-offs between the number of viruses detected and the depth and completeness of viral sequences emphasise that the most suitable sample type depends on the specific virus surveillance goal.

Replication analysis revealed important guidance for optimising sampling effort. When comparing the number of unique taxa collected from each swab replicate, a downward trend was observed following subsequent swabbing of the same area. For bacteria, only one additional unique taxon was detected from a second swab replicate, with no new taxa detected on the third. For fungi, however, swabbing replicates continued to detect additional unique taxa across each replicate, with 31 and 19 additional taxa detected in replicates two and three of the hive entrance swabs. Based on species accumulation curves, replication was sufficient for bacterial and animal communities, but further replication would be needed to recover additional fungal and plant diversity. The researchers recommend collecting two swab replicates per hive as sufficient for capturing taxonomic diversity from hive eDNA metabarcoding, while noting that sampling from additional hives is likely more beneficial than collecting more swab replicates from a single hive.

The effectiveness of hive entrance swabbing likely stems from the accumulation and persistence of eNA across the hive entrance and base. Hive debris typically accumulates at the base as a result of routine colony activities such as brood rearing and food storage. High bee traffic through the hive entrance facilitates the deposition of pollen, microbial spores, and other biological material from returning foragers, while passive air flow can draw eNA from the surrounding area into hives. The comparable and often better recovery of eNA from swabs over more direct sample types was somewhat surprising to the researchers, given that individual bees carry only a fraction of the total eNA and represent a narrower time window compared to honey and hive debris, which accumulate genetic material over longer periods. Each sample type captures a distinct temporal and ecological snapshot of the hive environment.

The study represents the most comprehensive comparison of hive eNA sample types to date and provides an improved sampling protocol that can be implemented for a range of biosecurity surveillance activities. Hive entrance swabbing offers clear logistical advantages: it is simple enough for non-specialists to collect, non-invasive for the ongoing persistence of hives, suitable for year-round sampling without seasonal constraints, and does not weaken colonies over time. The researchers suggest that leveraging established hive networks with this approach offers a highly scalable strategy for detecting honeybee pests and pathogens, plant pathogens, and surrounding vegetation across diverse landscapes. Engaging with beekeepers to capture hive eNA across urban and agricultural landscapes presents an exciting opportunity to support both hive health and environmental monitoring, one that can be strengthened through collaboration with citizen scientists and scaled across broad geographic areas with minimal impact on the hives themselves.

Subject of Research: Surface swabs outperform most traditional honeybee (Apis mellifera) hive samples for recovery of eDNA and eRNA

Article Title: Surface swabs outperform most traditional honeybee ( Apis mellifera ) hive samples for recovery of eDNA and eRNA

Article References: Hill, E., Milla, L., Encinas-Viso, F., O'Dwyer, J., Gooden, B., Hopper, M., & Roberts, J. (2025). Surface swabs outperform most traditional honeybee (Apis mellifera) hive samples for recovery of eDNA and eRNA. Metabarcoding and Metagenomics, 9, Article e165436. https://doi.org/10.3897/mbmg.9.165436

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.165436

Keywords: Surface, swabs, outperform, most, traditional, honeybee, Apis, mellifera, hive, samples, recovery, eDNA

The initiative was first proposed during the 26th International Conference on Subterranean Biology, held in Sardinia in September 2024. Nine months later, on June 5, 2025, GReG was formally launched through an online seminar led by Dr. Mattia Saccò, Dr. Michelle Guzik, and Dr. Kathryn Korbel. The event brought together 56 representative researchers and regulators from across the globe, marking the beginning of what organizers hope will become a unifying platform for advancing groundwater science through environmental DNA and genomics. The launch seminar featured guest speakers who shared insights from large-scale eDNA studies in oceans, discussed open and FAIR data practices for sharing eDNA information, explored taxon-free eDNA approaches in groundwater research, and examined how eDNA can be deployed to address global biodiversity targets.

Environmental DNA, broadly defined as the total pool of DNA isolated from environmental samples such as water or sediment, has emerged as a transformative tool for biodiversity assessment. By analyzing genetic material shed by organisms into their surroundings, researchers can detect species presence without directly observing or capturing them. The technique has proven successful at unveiling biodiversity patterns from regional to global scales and has been applied in taxonomy-free and functional approaches that bypass the need for traditional morphological identification. eDNA is also increasingly being combined with other large-scale monitoring tools, including satellite data and broader biodiversity datasets, to advance a rich and diverse research agenda across aquatic ecosystems.

In groundwater environments specifically, eDNA research is gaining momentum, with recent studies demonstrating its potential to characterize subterranean communities. However, the field lags behind other biomes such as oceans and rivers in terms of protocol validation, practical application, and the development of DNA assays tailored to subterranean conditions. This gap reflects the broader neglect of groundwater ecosystems in research and conservation, where many calls for action have gone unheeded. Groundwater species face escalating cumulative threats including climate change, land use change, contamination, over-extraction, heating, and salinization, among many others. Left unaddressed, these pressures compromise not only biodiversity but also the vast array of ecosystem services that groundwater environments quietly provide to both nature and human society.

GReG is conceived as a two-phase project designed to disrupt the current inertia in global groundwater conservation. Phase 1, running from June 2025 to June 2026, will involve fieldwork at more than 50 well-known sites across four continents, encompassing karst systems, alluvial aquifers, and anchialine caves. Researchers will apply standardized sampling and analytical protocols targeting biodiversity across the entire Tree of Life, ensuring that results are comparable across sites and regions. In parallel, a survey on methodological approaches in groundwater eDNA research has been circulated to all collaborators, aimed at understanding current practices and identifying limitations for eDNA analysis in groundwater environments. Further projected outputs include peer-reviewed papers on groundwater eDNA protocols and publicly available DNA barcode quality assessments for stygofauna, the specialized animals that inhabit groundwater.

Phase 2, beginning in July 2026, will build on the lessons learned during the first phase to expand the project to the Global South and other under-investigated areas of the world through participatory science approaches. The initiative’s organizers have issued a public call to researchers and stakeholders from these regions, with the ambition of widening novel groundwater-based research beyond well-known global biodiversity hotspots. The collaborative effort is supported by partners including Biodiversa+ projects such as DarCO and Sub-BioMon, research laboratories including the TrEnD Lab at Curtin University and the Altermatt Lab, and global datasets such as Stygofauna Mundi, which catalogues the diversity of groundwater fauna worldwide.

The outcomes of this collaborative effort are expected to include scientific publications, data sharing following FAIR principles—ensuring that findings are Findable, Accessible, Interoperable, and Reusable—workshops, and media articles, all with the objective of advancing subterranean aquatic eDNA research. Beyond the scientific outputs, GReG also aims to raise awareness about the importance of groundwater ecosystems, a notoriously difficult task for environments that remain out of sight and out of mind for most of the public and policymakers alike. By bringing together researchers, regulators, industry stakeholders, and citizens, the initiative seeks to build the interdisciplinary and inclusive coalition needed to translate scientific knowledge into effective conservation action.

The development of conservation strategies for groundwater and groundwater-dependent ecosystems—those that rely on groundwater for some or all of their water needs—requires an improved understanding of subterranean ecological dynamics, particularly under ongoing climatic changes. Current global knowledge on groundwater biodiversity and ecological dynamics remains far from complete, hindering effective assessment and conservation efforts. GReG’s founders argue that the key to preserving unique subterranean biota while using groundwater resources sustainably is not yet lost; what is needed is the right team of researchers, regulators, stakeholders, and citizens working together to develop the biomonitoring tools and conservation guidelines that these hidden ecosystems urgently require.

As the first coordinated global effort to apply eDNA across groundwater ecosystems, GReG represents an ambitious but vital step toward securing the future of the planet’s least visible biodiversity. Its success will depend on sustained collaboration across continents, disciplines, and sectors, and on the willingness of the scientific community and policymakers to finally give groundwater the attention it has long been denied. For an environment that supplies a substantial share of the world’s drinking water and supports irreplaceable evolutionary heritage, the stakes could hardly be higher.

Subject of Research: Environmental DNA-based global research initiative for groundwater biodiversity conservation

Article Title: Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG)

Article References: Saccò, M., Elmasri, A., Tawal, M., Alther, R., Altermatt, F., Martínez, A., Mammola, S., Zagmajster, M., Saenz-Agudelo, P., Takahashi, M., Thornhill, J., Andouche, A., Baratti, M., Bilal, M., Bilandžija, H., Bolpagni, R., Boulamail, S., Celico, F., Chauveau, C. A., … Guzik, M. T. (2025). Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG). Subterranean Biology, 53, 31-40. https://doi.org/10.3897/subtbiol.53.165710

Image Credits: AI Generated

DOI: 10.3897/subtbiol.53.165710

Keywords: environmental DNA, groundwater, subterranean biodiversity, eDNA, conservation, stygofauna, aquifers, GReG, karst, anchialine caves, FAIR data, Global South

The blue crab is native to the western Atlantic Ocean and most likely entered the Mediterranean during the 1940s through ballast water discharge from transoceanic shipping. Since then its populations have expanded across a wide range of coastal habitats, with a marked acceleration since approximately 2017. The species is an opportunistic predator with broad trophic flexibility, exploiting fish, invertebrates, polychaetes, macrophytes and especially bivalves, and documented declines in native bivalve stocks have followed its arrival, from oysters in Spain’s Ebro Delta to the aquaculture of the Manila clam in Italy’s Po Delta. Females can produce up to four million eggs per spawning event, and the crab is euryhaline, tolerating wide ranges of salinity and temperature, which allows it to colonize coastal waters, estuaries and lagoons with equal ease.

These traits combine to make early detection both difficult and urgent. Under Regulation (EU) No 1143/2014, early detection means confirming an invasive alien species before it becomes widely established, yet conventional surveys based on trapping and visual census frequently miss low-density invasions. In the Ebro Delta, roughly five years elapsed between the first detection of the blue crab and the onset of exponential population growth. Notably, the species is not currently listed among the invasive alien species of Union concern, and recent analyses suggest its Adriatic expansion is being facilitated by rising sea surface temperatures, adding urgency to calls for harmonized surveillance and coordinated management across Member States.

Environmental DNA metabarcoding offers a molecular alternative. By extracting DNA directly from water or sediment, amplifying universal genetic markers such as COI with degenerate primers, and applying high-throughput sequencing, researchers can identify entire biological communities from a single sample. But the approach has well-known constraints. Incomplete reference databases limit taxonomic resolution in marine and transitional ecosystems, and because universal primers amplify DNA from many taxa simultaneously, DNA from a rare target species competes with DNA from all co-occurring organisms during PCR. Low-abundance species may therefore be underrepresented in sequencing output or excluded during bioinformatic filtering, undermining the very sensitivity needed for early detection of invaders.

Targeted eDNA assays sidestep this competition by using highly specific primers that selectively amplify DNA from a single species. The Salento team began by downloading five COI sequences representative of Mediterranean blue crab populations from NCBI GenBank and establishing the forward primer position with Primer-BLAST. To minimize cross-reactivity, they compiled a list of roughly 1300 aquatic animal species co-occurring with the crab in Mediterranean coastal and transitional ecosystems, retrieving Folmer-region COI sequences for about 600 of them and testing the candidate primer in silico against this panel. The reverse primer was designed manually outside the Folmer region, about 500 base pairs downstream, to yield an amplicon optimal for both Sanger sequencing and next-generation sequencing while ensuring reliable species-level identification.

Only the primer pair predicted to amplify exclusively Callinectes sapidus was retained. The final primers, named CSapFor and CSapRev, were then validated experimentally. Genomic DNA extracted from blue crab muscle tissue produced a single amplicon of the expected 464 base pairs, while DNA from two non-target control species, the brown shrimp Penaeus aztecus and the Mediterranean mussel Mytilus galloprovincialis, generated no visible bands under the tested PCR conditions. Sanger sequencing of the crab amplicon returned a BLAST match exclusively with Callinectes sapidus sequences, with 100% sequence identity, confirming that the amplified fragment corresponds to the intended target region.

The researchers then applied the assay to environmental samples. One-liter surface water samples were collected from the Aquatina di Frigole lagoon in southeastern Italy, the Neretva River Delta in Croatia, the San Benedetto del Tronto coast and Tronto River Delta in central Italy, and the Venice Lagoon, yielding 22 eDNA samples in total. Each replicate was pre-filtered through a 0.5-millimeter mesh to remove particulates and PCR inhibitors, then filtered through 0.45-micrometer cellulose filters from which DNA was extracted. High-throughput sequencing of the COI amplicons on an AVITI platform generated 10,773,406 raw reads, averaging about 500,000 reads per sample. Every operational taxonomic unit assigned at the species level was identified as Callinectes sapidus, and the most abundant OTU was detected across all pilot areas, demonstrating the assay’s ability to identify the species even at low DNA concentrations.

Because similarity-based classification alone cannot assess the evolutionary coherence of identified sequences, the team added independent phylogenetic validation. They built a maximum-likelihood tree from 96 blue crab amplicon sequence variants together with reference COI sequences representing the diversity of the genus Callinectes and a mud crab sequence as an outgroup. The topology resolved two well-supported lineages among the reference taxa, consistent with previously published molecular phylogenies of the genus, and all metabarcoding-derived variants clustered coherently within the Callinectes sapidus lineage, clearly separated from congeneric species. The researchers caution that the more than 2500 variants should not be read as extensive evolutionary diversification; amplicon sequence variants preserve even single-base polymorphisms, so many represent closely related mitochondrial haplotypes differing by only one or a few nucleotides, as reflected in the short branch lengths within the crab clade.

A haplotype network analysis added a phylogeographic dimension. The network resolved a star-like topology anchored by a predominant central hub spanning all Adriatic pilot areas and Mediterranean reference sites, with single-mutation tip haplotypes radiating outward. The dominant Mediterranean hub appears directly connected to reference sequences from Mexico, supporting the hypothesis that the species was introduced from the Gulf of Mexico via ballast water transport, with the broad distribution of the primary haplotype across all Adriatic locations reflecting high gene flow driven by prolonged pelagic larval drift and the strong oceanographic connectivity of the Adriatic Sea. Distinct spatial partitioning emerged, with high haplotype richness in the Neretva River Delta and Aquatina di Frigole Lagoon and a subset of variants apparently localized in the Venice Lagoon, hinting at localized demographic processes and ongoing in situ microdiversification after settlement.

The study represents one of the few experimental demonstrations confirming the feasibility of detecting an invasive species through targeted eDNA analysis, complementing earlier probe-based qPCR methods for the blue crab. Previous analyses of the same Adriatic pilot areas using the widely adopted degenerate primer pair mCOIintF/dgHCO2198 did not consistently detect the species, whereas the species-specific primers succeeded across all sites, underscoring the sensitivity gain that targeted design delivers. Beyond early detection, the approach yields genetic data that can reconstruct invasion dynamics at the molecular level, identifying haplotypes originating in the native range or following introduction into the Mediterranean. As rising temperatures continue to favor the establishment and spread of this invader, the researchers argue that integrating molecular detection methods into invasive species management offers a powerful means to detect and track invaders even in the absence of physical specimens, ultimately informing the targeted control measures needed to protect native Mediterranean coastal ecosystems.

Subject of Research: Development and validation of a targeted environmental DNA assay for early detection of the invasive blue crab Callinectes sapidus in Adriatic coastal-marine ecosystems

Article Title: Targeted eDNA Survey Enables Consistent Detection of the Invasive Blue Crab in Trans‐Border Adriatic Coastal‐Marine Ecosystems

Article References: De Donno, M. D., Zangaro, F., Arnosti, P., Bergamin, S., Boban, J., Bozzetti, M. P., Cazzetta, A., De Pittà, C., Dessanai, D., Drigo, F., Julius, O. T., Ključe, L., Lušić, J., Marcucci, F., Massaro, R., Morelli, V., Moro, I., Nazlić, N., Pellizzato, M., … Pinna, M. (2026). Targeted eDNA Survey Enables Consistent Detection of the Invasive Blue Crab in Trans‐Border Adriatic Coastal‐Marine Ecosystems. Ecology and Evolution, 16(10), Article e74462. https://doi.org/10.1002/ece3.74462

Image Credits: AI Generated

DOI: 10.1002/ece3.74462

Keywords: Targeted, eDNA, Survey, Enables, Consistent, Detection, Invasive, Blue, Crab, Trans, Border, Adriatic