Avian schistosomes (AS) are trematodes of significant medical importance as the causative agents of cercarial dermatitis, a re-emerging waterborne human disease. Their monitoring has traditionally relied on examination of snail hosts; however, this approach is time-consuming, requires taxonomic expertise and often underestimates their diversity due to low prevalence of infection. Although environmental DNA (eDNA)-based methods have shown promise in detecting AS, most studies have employed species-specific assays that restrict the discovery of novel genetic lineages. This study integrated three complementary methodologies to comprehensively assess AS diversity in Central Europe: (i) metabarcoding of environmental water samples from various localities; (ii) metabarcoding of naturally infected snail hosts to detect pre-patent stages and (iii) standard parasitological examination and collection of cercariae released from snail hosts. In total, 34 species/genetic lineages were detected at 19 localities. Of these, three species were identified by all three techniques, two lineages by both water sample and snail tissue metabarcoding; 17 exclusively through water sample metabarcoding, 10 only in snail tissues and two solely by the standard method. Notably, metabarcoding of environmental water samples was the most powerful tool for capturing most of the AS diversity, while snail tissue metabarcoding uncovered early pre-patent infections and additional unique lineages not detected in water. These findings underscore the critical value of a multi-method approach to minimise detection bias associated with any single technique.
Environmental DNA (eDNA) is rapidly becoming a valuable tool for conducting biodiversity research, including studies on marine vertebrates, and metabarcoding of eDNA enables the characterization of biological communities. However, methodological variation across workflow stages can influence results, highlighting the need for standardized and accessible protocols. In this study, two eDNA extraction strategies were evaluated, the performance of two DNA polymerases differing in proofreading capacity was tested, and the Marine Vertebrate eDNA Metabarcoding bioinformatics pipeline (MVeM)—an open-source, adaptable, and reproducible workflow for sequence processing and taxonomic assignment–was developed. For extraction, the standard DNeasy PowerWater Sterivex protocol was applied, and a preliminary bead-beating homogenization step performed on filter material prior to the PowerWater protocol was additionally tested, as was the recovery of free extracellular DNA in controlled seawater samples. Direct extraction from intact filters yielded higher DNA concentrations and amplicon sequence variant (ASV) richness than the bead-beating homogenization method, whereas both approaches effectively captured marine vertebrate diversity. The recovery of free extracellular DNA remained low with both extraction methods. During library preparation and sequencing, the non-proofreading polymerase generated more cetacean ASV reads in mock communities, suggesting that it is a cost-effective option for monitoring this group, whereas the proofreading enzyme improved the amplification of fish taxa. The MVeM pipeline integrates stringent sequence filtering, the Lowest Common Ancestor approach for ambiguous matches, a geographic exclusion list, and contamination control, enabling accurate, biologically realistic, and reproducible taxonomic assignments for marine vertebrate eDNA. Overall, combining appropriate extraction strategies, polymerase selection, and a robust bioinformatics framework provides a reliable, adaptable, and accessible approach for marine vertebrate biodiversity monitoring. Graphical abstract :
Microbial communities influence key processes in oil reservoirs, including souring, corrosion, and hydrocarbon degradation, so characterizing them is important for reservoir management. Reliable characterization depends on obtaining a sample that represents the community living in the subsurface. As nucleic acids are difficult to recover from oil, produced water, the aqueous phase separated from reservoir fluids, is widely used as a convenient proxy for analyzing the reservoir microbiome. However, whether produced water faithfully represents that community, to our knowledge, has never been tested with paired samples. We addressed this question using 16S rRNA gene amplicon and metagenomic sequencing of reservoir fluids and produced water collected from the same production wells across four North Sea oil fields. The two sample types harbored markedly different microbial communities and functional profiles. This difference persisted after accounting for reservoir identity, and it was larger than the differences between reservoirs. Produced water was less diverse, more uniform across fields, and dominated by aerobic genera associated with seawater and aquifers. Reservoir fluids, in contrast, retained the thermophilic, halophilic, and anaerobic taxa characteristic of the deep subsurface. Only 41% of reservoir-fluid genera and 21% of their amplicon sequence variants were also detected in the paired produced water, indicating overlapping broad taxa but largely distinct populations. Notably, the putatively native reservoir community remained detectable in reservoir fluids even after decades of production, while produced water offered only an incomplete, directionally biased view. Reservoir fluids should therefore be sampled wherever feasible to characterize the in-situ microbiome.
Environmental DNA (eDNA) analysis has emerged as a transformative tool for biodiversity assessment and environmental monitoring, offering a non-invasive, cost-effective approach to detecting species and characterizing ecological communities. Despite growing adoption by researchers, regulatory agencies, and industry, the widespread implementation of eDNA methods continues to face challenges, including inconsistent protocols across laboratories and the absence of internationally recognized standards. These barriers have slowed the integration of eDNA into routine monitoring programs and other operational settings, particularly in regulatory contexts. Standardization is widely recognized as an essential prerequisite for building confidence among end users and enabling meaningful comparison of results across studies and jurisdictions. While standards development efforts have begun to emerge at national and regional scales, the lack of international coordination risks producing fragmented or contradictory guidelines that could hinder rather than advance the field. Here, we introduce the International eDNA Standardization Task Force (iESTF; https://iestf.global), an initiative established to facilitate and accelerate the development of inclusive international standards for eDNA-based biodiversity monitoring. The iESTF aims to identify opportunities for new work item proposals within the International Organization for Standardization (ISO) framework, catalog existing standards and best practices, and ensure that standards development is accessible to practitioners in nations historically underrepresented in eDNA research. We outline some key challenges facing eDNA standardization, describe the structure and goals of the iESTF, and present a vision that includes the formation of a truly inclusive international community of practice, the establishment of pipelines for fast-tracking the development of international standards, and support for critical activities such as cross-regional standards testing and validation. The iESTF represents a critical step toward ensuring that eDNA methods achieve their full potential as reliable, globally comparable tools for biodiversity monitoring.
Freshwater biodiversity underpins ecosystem functioning but is rapidly declining due to urbanization and habitat modification, particularly in Southeast Asia where accurate species inventories remain limited. Integrative taxonomy, combining morphological and molecular approaches, provides a robust framework for generating standardized biodiversity data and biomonitoring assessments. We assessed mayfly (Ephemeroptera) biodiversity across a natural-to-urbanized gradient in Hong Kong using nymph morphology and cytochrome c oxidase subunit I (COI) barcoding to establish the first regional DNA barcode reference library. Our integrated taxonomy efforts resulted in 45 unique morphospecies (i.e. gamma-diversity). Subsequently, the Hong Kong mayfly barcode database includes 146 barcode sequences representing 53 Barcode Index Numbers (BINs), of which 67.3% were newly registered in the Barcode of Life Data System (BOLD), including seven putative undescribed lineages and four new regional records. Additionally, we found that mayfly richness declined significantly with increasing urban intensity (β = –0.308, p = 0.007) and conductivity (β = –0.427, p = 0.002), with elevation showing a marginal effect. Indicator species analysis identified Choroterpes elliptica HK01 as potentially indicative of natural sites and Tenuibaetis pseudofrequentus and Caenis lubrica likely corresponding with peri-urban habitats, while no taxa were indicative of urbanized sites. Our results demonstrate both the substantial mayfly diversity in Hong Kong and highlight the detrimental impacts of urbanization on freshwater assemblages. Critically, the barcode library presented here supports future DNA-based biomonitoring applications (e.g. eDNA metabarcoding), providing an important resource for rapid biodiversity assessment of critical habitats and helping to ensure long-term ecosystem health and stability.
