Metabarcoding Approach Research Articles

Limited Variation in Bacterial Communities of Scaphoideus titanus (Hemiptera: Cicadellidae) Across European Populations and Different Life Stages

The Nearctic leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae) is the primary vector of ‘Candidatus Phytoplasma vitis’, the causative agent of Flavescence doreé in Europe. Although microorganisms play an important role in the ecology and behavior of insects, knowledge about the interaction between S. titanus and microbes is limited. In this study, we employed an amplicon metabarcoding approach for profiling the V4 region of the 16S rRNA gene to characterize the bacterial communities of S. titanus across several populations from four European localities. Additionally, we investigated changes in bacterial communities between nymphal and adult stages. In total, we identified 7,472 amplicon sequence variants (ASVs) in adults from the European populations. At the genus level, ‘Candidatus Karelsulcia’ and ‘Candidatus Cardinium’ were the most abundant genera, with both being present in every individual. While we found significant changes in the microbial composition of S. titanus across different European populations, no significant differences were observed between nymphal and adult stages. Our study reveals new insights into the microbial composition of S. titanus and highlights the role of geography in influencing its bacterial community.

Prey diversity in the deep ocean: metabarcoding feeding ecology of the commercially important queen snapper in the US Caribbean

The queen snapper, Etelis oculatus, is experiencing a growing fishery presence across the wider Caribbean, yet our understanding of its distribution, biology, and ecology remains limited. To address this gap, we investigated the feeding ecology of this demersal deep-water snapper using two molecular approaches: single-species barcoding and multispecies metabarcoding. Between November 2019 and July 2020, we collected 157 queen snapper from seven locations along the west coast of Puerto Rico. Our analysis identified a diverse array of prey items in the stomachs, comprising 61 species from 31 families, 18 orders, and 38 genera. Our findings suggest that E. oculatus is a large carnivore, primarily preying on squids, shrimps, and deep-water fishes. Notably, common fish prey included Diaphus brachycephalus, D. dumerilii, Dasyscopelus selenops, Coccorella atlantica, Sigmops elongatus, and Zaphotias pedaliotus. In contrast, prevalent invertebrate prey consisted of Abralia veranyi, Doryteuthis pealeii, Abralia redfieldi, Oplophorus gracilirostris, and Systellaspis debilis. Although our data suggest potential variation in diet composition across locations, definitive conclusions remain elusive. However, we observed heightened prey richness at seamount Pichincho, possibly attributable to the high structural complexity of the karst terrain on the seafloor. Additionally, we noted variation in prey species composition across size classes, with certain species more prevalent in larger or smaller queen snapper. Given the challenge of examining the prey of deep-water fish species, our study showcases the utility of both single-species barcoding and multispecies metabarcoding methodologies in characterizing the dietary range of queen snapper and similar demersal deep-water carnivorous fishes. The multispecies metabarcoding approach, in particular, offers a rapid, comprehensive, and effective means of identifying prey species. As the commercial value of the queen snapper continues to rise, our investigation into its feeding behavior provides critical baseline information for future species management efforts. By enhancing our understanding of its ecological dynamics, we aim to contribute to informed conservation and sustainable fisheries practices in the region.

What Quality Suffices for Nanopore Metabarcoding? Reconsidering Methodology and Ectomycorrhizae in Decaying Fagus sylvatica Bark as Case Study.

Nanopore raw read accuracy has improved to over 99%, making it a potential tool for metabarcoding. For broad adoption, guidelines on quality filtering are needed to ensure reliable taxonomic unit recovery. This study aims to provide those guidelines for a fungal metabarcoding context and to apply them to a case study of ectomycorrhizae in the decaying bark of Fagus sylvatica. We introduce the eNano pipeline to test two standard metabarcoding approaches: (1) Reference-based mapping leveraging UNITE's species hypothesis system (SH approach); (2) Constructing 98% OTUs (OTU approach). Our results demonstrate that both approaches are effective with Nanopore data. When using a reference database, we recommend strict mapping criteria rather than Phred-based filtering. Leveraging the SH-system further enhances reproducibility and facilitates cross-study communication. For the 98% OTUs, filtering reads at ≥Q25 is recommended. Our case study reveals that the decay gradient is a primary determinant of community composition and that specific mycorrhizal fungi colonize decaying bark. Complementing our metabarcoding results with root tip morphotypification, we identify Laccaria amethystina and Tomentella sublilacina as key ectomycorrhizae of saplings on decaying logs. These findings demonstrate that Nanopore sequencing can provide valuable ecological insights and support its broader use in fungal metabarcoding as read quality continues to improve.

Yeast mycobiome of fruit and vegetable biowastes revealed by culture-dependent and metabarcoding approaches: Screening for the production of succinic acid and single cell oils

Fruit and vegetable by-products and agro-residues from food processing industries and households negatively impact the environment, economy, and society. Yeasts are metabolically efficient at converting food wastes into value-added products such as biopeptides, single-cell oils, single-cell proteins, and organic acids. This work explores the valorization of twelve fruit and vegetable-derived wastes as inexpensive substrates for obtaining high-value products and metabolically efficient yeasts. Culture-dependent and metabarcoding methods provided a comprehensive overview of the yeast mycobiome, revealing 33 common species. From a biobank of 252 isolates obtained from the twelve biowastes, the 31 most promising isolates were evaluated using a synthetic medium that mimicked fruit and vegetable waste. The results showed that the metabolism was mainly directed towards the Krebs cycle, with lower production of single-cell oil compared to organic acids. Kazachstania humilis stood out from the general metabolic heterogeneity, producing 0.9 g/L of succinic acid and 1.4 g/L of acetic acid. Our results demonstrate that these natural isolates can produce high-value compounds at levels comparable to those obtained under optimal conditions for yeast growth.

Harmful Microalgae Exhibit Broad Environmental Adaptability in High-Salinity Area Across the Dafengjiang River Estuary.

Harmful algal blooms (HABs) often occur in estuaries due to their unique environmental heterogeneity, posing significant environmental and human health risks. However, there is limited understanding of the community composition and community-level change points (thresholds) of harmful microalgae in subtropical estuaries. This study explored harmful microalgae community structure and thresholds in the Dafengjiang River estuary using a metabarcoding approach. The results revealed 63 harmful microalgae species, and major species included Guinardia flaccida, Prorocentrum cordatum, Thalassiosira punctigera, Pseudo-nitzschia galaxiae and T. gravida. Nonparametric change-point analysis and threshold indicator taxa analysis (TITAN) showed threshold responses of harmful microalgae community structure to ammonium (57.5-60 μg·L-1), total phosphorus (27.8-28.5 μg·L-1) and dissolved inorganic phosphorus (14.5-28 μg·L-1) along the salinity gradient. Wider environmental thresholds were also found in hypersaline areas. Additionally, Pyrodinium bahamense, Pfiesteria piscicida, Skeletonema tropicum and T. punctigera were sensitive to environmental changes and thus could be used as bioindicators. Overall, our study unveiled diverse abrupt transitions of harmful microalgal communities, providing a risk assessment for human health and ecological safety in subtropical estuary ecosystems.

Influence of Biofloc Technology and Continuous Flow Systems on Aquatic Microbiota and Water Quality in Japanese Eel Aquaculture

Biofloc technology (BFT) systems heavily rely on microbiota to mitigate ammonia toxicity and manage essential nutrient cycling. Understanding the diversity and functional role of microbiota within BFT-applied aquaculture systems is crucial for ensuring sustainable operations. Though some studies exist on BFT microbiota, research on microbial differences in Japanese eel aquaculture is still limited, hindering the wider application of BFT systems. In this study, we analyzed the characteristics of water quality factors and microbiota in Japanese eel (Anguilla japonica) breeding water, applying the BFT system. Using a metabarcoding approach, the diversity and community structure of aquatic microbiota were compared between BFT and continuous flow (CF) systems. The pH was significantly higher in CF water, while total ammonia nitrogen (TAN) and nitrite (NO2−-N) was higher in BFT water. Alpha diversity was significantly higher in BFT compared to CF systems, and it was correlated significantly with pH and TAN. In both BFT and CF water, the phyla Proteobacteria and Bacteroidota were found to be the most abundant. In the BFT water, a diverse array of bacterial taxa, including BFT-specific clades, were consistently present, while the microbiota in CF water was more variable and contained fewer specific taxa. In addition, bacterial functions related to nitrate reduction, sulfur compound oxidation, and chitinolysis were significantly more abundant in BFT than in CF systems. These findings highlight differences in water quality and microbiota between aquaculture systems, which can inform future research on the use of BFT for sustainable fish farming.

Decline of beech trees in a Mediterranean forest is associated with high rhizosphere oomycete diversity

In the last decades, tree decline in European beech forests has been related to extreme climatic events and with the activity of parasitic oomycetes such as Phytophthora species. In Spain, little is known about the association of beech decline and soil oomycete composition. We hypothesized that the weakening of beech trees is associated with the activity and proliferation of parasitic oomycetes. We studied the rhizosphere oomycete community of large beech trees located in one of the southernmost beech forests in Europe. From soil samples collected in the rhizosphere, we used a metabarcoding approach to explore the oomycete community associated to asymptomatic and declining trees. We also studied fine root parameters in both groups of trees. A total of 99 oomycete amplicon sequence variants (ASVs) were detected. Higher diversity and richness of oomycetes were observed in declining than in asymptomatic trees. The oomycete composition also differed between the two groups of trees. Declining trees showed lower fine root biomass and root density than asymptomatic trees, and root density was negatively correlated with the abundance of Pythium ASV counts. The genus Phytophthora, associated with beech decline in central Europe, was underrepresented in the oomycete community. The results suggest that decline-associated processes in beech trees are related with fine root weakening and loss which probably facilitates the entrance and colonization of opportunistic oomycete microbes. Although members in the genus Pythium do not seem to be a primary factor on beech decline, they probably contribute to the chronic decline of Fagus sylvatica trees.

Unique Features of Extremely Halophilic Microbiota Inhabiting Solar Saltworks Fields of Vietnam.

The artificial solar saltworks fields of Hon Khoi are important industrial and biodiversity resources in southern Vietnam. Most hypersaline environments in this area are characterized by saturated salinity, nearly neutral pH, intense ultraviolet radiation, elevated temperatures and fast desiccation processes. However, the extremely halophilic prokaryotic communities associated with these stressful environments remain uninvestigated. To fill this gap, a metabarcoding approach was conducted to characterize these communities by comparing them with solar salterns in northern Vietnam as well as with the Italian salterns of Motya and Trapani. Sequencing analyses revealed that the multiple reuses of crystallization ponds apparently create significant perturbations and structural instability in prokaryotic consortia. However, some interesting features were noticed when we examined the diversity of ultra-small prokaryotes belonging to Patescibacteria and DPANN Archaea. Surprisingly, we found at least five deeply branched clades, two from Patescibacteria and three from DPANN Archaea, which seem to be quite specific to the Hon Khoi saltworks field ecosystem and can be considered as a part of biogeographical connotation. Further studies are needed to characterize these uncultivated taxa, to isolate and cultivate them, which will allow us to elucidate their ecological role in these hypersaline habitats and to explore their biotechnological and biomedical potential.

Unveiling the Gut Microbiota of Pig-Tailed Macaque (Macaca nemestrina) in Selected Habitats in Malaysia.

The gut microbiota plays an important role in primates, which may be associated with their habitat. In Malaysia, pig-tailed macaques (Macaca nemestrina) live in different habitat environments and have traditionally been used for coconut plucking for more than a century. There is currently no information regarding the gut microbiota of this macaque in Malaysia. To address this oversight, this study employed a fecal metabarcoding approach to determine the gut microbiota composition of pig-tailed macaques and establish how these microbial communities correspond with the macaque external environments of residential area, forest edge, and fragmented forest. To determine this connection, 300 paired-end sequences of 16S rRNA were amplified and sequenced using the MiSeq platform. In the pig-tailed macaque fecal samples, we identified 17 phyla, 40 orders, 52 families, 101 genera, and 139 species of bacteria. The most prevalent bacterial families in the gut of pig-tailed macaques were Firmicutes (6.31%) and Proteobacteria (0.69%). Our analysis did not identify a significant difference between the type of environmental habitat and the gut microbiota composition of these macaques. There was great variation in the population richness and bacterial community structure. The abundance of Firmicutes and Proteobacteria helps this macaque digest food more easily while maintaining a healthy gut microbiota diversity. Exploring the gut microbiota provides an initial effort to support pig-tailed macaque conservation in the future.

Evaluating the quantitative performance of environmental DNA metabarcoding for freshwater zooplankton community: a case study in Lake Biwa, Japan.

Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future.

DNA Metabarcoding Approach as a Potential Tool for Supporting Official Food Control Programs: A Case Study.

Food authentication significantly impacts consumer health and the credibility of Food Business Operators (FBOs). As European regulations mandate the verification of food authenticity and supply chain integrity, competent authorities require access to innovative analytical methods to identify and prevent food fraud. This study utilizes the DNA metabarcoding approach on meat preparations, sampled during an official control activity. It assesses animal and plant composition by amplifying DNA fragments of the 12S rRNA and trnL (UAA) genes, respectively. The results not only confirmed the declared species but also revealed undeclared and unexpected taxa in products labelled as containing a single animal species and various unspecified plant species. Notable findings such as the presence of Murinae, Sus scrofa, Ovis aries, and Pisum sativum could raise public health concerns, compromise consumer choices made for ethical or religious reasons, and reflect the hygienic conditions of the processing plant. This study demonstrates that the DNA metabarcoding approach looks to be a promising support tool for official control authorities to ensure food authenticity and safety, and to develop risk profiles along the supply chain.

Performance of DNA metabarcoding, standard barcoding and morphological approaches in the identification of insect biodiversity.

For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single-specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment.

A comparative analysis of eDNA metabarcoding and field surveys: Exploring freshwater plant communities in rivers

While environmental DNA (eDNA) metabarcoding holds promise as a holistic approach to assess vegetation changes and community composition across diverse spatial and temporal scales, systematic investigations of its efficacy compared to conventional field surveys remain scarce in the literature. The present study explores the differences in plant diversity recovered from field surveys and captured with a multi-marker eDNA metabarcoding approach (two nrDNA ITS1 and ITS2, and two cpDNA rbcL and trnL) from river water samples. The eDNA metabarcoding approach retrieved 46 aquatic plants (hydrophytes and helophytes) and 245 terrestrial plants, compared to 24 and 127 species identified from field surveys. On average, eDNA samples collected immediately downstream of the survey sites recovered 43 % and 39 % of the aquatic and terrestrial species observed, respectively. Discrepancies were explained by differences in taxonomic resolution, the stochasticity of the retrieval of rare and elusive species, and the presence of reference sequences. We found a significant positive correlation between spatial and community distances at scales ranging from 2 to 9 km and identified turnover as the driving force of these differences. Metabarcoding demonstrated sensitivity to community changes and both approaches converge on a similar community structure. Interestingly, eDNA samples collected immediately upstream of the survey sites exhibited significant species overlap with the downstream samples (c. 100 m apart). Overall, our results demonstrate that within-site species mismatches between the methods are nonnegligible, and they question the use of eDNA for generating complete species lists at scales comparable to our field surveys (< 100-m transects). However, with adequate sampling and a multi-marker metabarcoding approach, eDNA has the potential to approximate catchment gamma diversity with less sampling effort than conventional surveys.

Impact of a coastal protection measure on sandy-beach meiofauna at Ahrenshoop (Baltic Sea, Germany): results from metabarcoding and morphological approaches are similar

Sand nourishment is a common and often applied coastal protection measure along the beaches of the German Baltic Sea coast. At the tourist beach of Ahrenshoop, large-scale sand nourishments are completed every five years along a 4-km stretch. The nourishment in question was executed between the end of October 2021 and mid-February 2022. Meiofaunal communities at the beach-water interface were monitored at six stations before (September 2021) and for one year after the impact (March and September 2022, March 2023). To assess the effects of sand nourishment on meiofauna communities, two complementary approaches were employed: whole community metabarcoding and morphological determination and counting of the animals. Both methods produced consistent results regarding community response to disturbance and subsequent recovery. During the period from autumn 2021 to spring 2023, drastic changes in the composition of communities were observed. Directly after the impact, the taxa Acari and Annelida nearly disappeared, abundances of Copepoda decreased substantially, and Platyhelminthes increased notably. The fact that the studied beach held a low diversity may have facilitated that metabarcoding and morphological determination followed the same trend. After initial studies comprising both methods, efficient assessments using only metabarcoding may be considered for comparable sites and situations.

From ctenophores to scyphozoans: parasitic spillover of a burrowing sea anemone

Most host-parasite associations are explained by phylogenetically conservative capabilities for host utilization, and therefore parasite switches between distantly related hosts are rare. Here we report the first evidence of a parasitic spillover of the burrowing sea anemone Edwardsiella carnea from the invasive ctenophore Mnemiopsis leidyi to two scyphozoan hosts: the native Mediterranean barrel jellyfish Rhizostoma pulmo and the invasive Indo-Pacific nomad jellyfish Rhopilema nomadica, collected from the Eastern Mediterranean Sea. Edwardsiella carnea planulae found in these jellyfish were identified using molecular analyses of the mitochondrial 16S and nuclear 18S rRNA genes. Overall, 93 planulae were found on tentacles, oral arms, and inside of the gastrovascular canals of the scyphomedusae, whereas no infection was observed in co-occurring ctenophores. DNA metabarcoding approach indicated seasonal presence of Edwardsiella sp. in the Eastern Mediterranean mesozooplankton, coinciding with jellyfish blooms in the region. Our findings suggest a non-specific parasitic relationship between Edwardsiella carnea and various gelatinous hosts based on shared functionality rather than evolutionary history, potentially driven by shifts in host availability due to jellyfish blooms. This spillover raises questions about the ecological impacts of parasitism on native and invasive scyphozoan hosts and the potential role of Edwardsiella in controlling their populations.

Rhizobacterial diversity of Portuguese olive cultivars in the Douro valley and their potential as plant growth promoters.

This study investigated the bacterial communities in the rhizosphere of two traditional Portuguese olive cultivars, Cobrançosa and Negrinha de Freixo, in relation to soil properties. Additionally, we aimed to isolate and identify bacteria with potential for biocontrol and other plant growth-promoting traits from these rhizosphere communities. Bacterial communities in the olive rhizosphere were investigated using a metabarcoding approach and the soil physicochemical properties of the olive groves were also analyzed. Higher bacterial richness was associated with Negrinha de Freixo growing in soil with high organic matter content and water-holding capacity. In contrast, the soils of the Cobrançosa grove presented higher pH and electric conductivity. Negrinha de Freixo rhizosphere was enriched with ASVs (Amplicon Sequence Variants) belonging to Bacillus, Gaiella, Acidothermus, Bradyrhizobium, and uncultured Xanthobacteraceae. On the other hand, the Cobrançosa rhizosphere was characterized by higher relative abundance of Streptomyces and Sphingomonas. Bacterial isolation from the rhizosphere and screening for plant growth-promoting activities were also performed. Six bacteria strains, predominantly Bacillus isolated from Negrinha de Freixo, demonstrated antagonistic activities against the olive fungal pathogen Colletotrichum gloeosporoides and other plant growth promotion (PGP) traits. Our findings demonstrate that the structure of rhizosphere bacterial communities associated with olive trees is shaped by both plant cultivar and soil-related factors. The higher number of bacterial species in the rhizosphere of Negrinha de Freixo was related to a higher organic matter content and a greater abundance of isolates with plant growth promotion traits, particularly Bacillus strains.

Structural variability of protist assemblages in surface sediments across Italian Mediterranean marine subregions

Marine sediments host heterogeneous protist communities consisting of both living benthic microorganisms and planktonic resting stages. Despite their key functions in marine ecosystem processes and biogeochemical cycles, their structure and dynamics are largely unknown. In the present study, with a spatially intensive sampling design we investigated benthic protist diversity and function of surface sediment samples from three subregions of the Mediterranean Sea, through an environmental DNA metabarcoding approach targeting the 18S V4 region of rRNA gene. Protists were characterized at the taxonomic level and trophic function, both in terms of alpha diversity and community composition, testing for potential differences among marine subregions and bathymetric groups. Overall, Alveolata and Stramenopiles were the two divisions that dominated the communities. These dominant groups exhibited significant differences among the three Mediterranean subregions in the alpha diversity estimates based on the detected ASVs, for all computed indices (ASV richness, Shannon and Simpson indices). Protist communities were also found to be significantly different in terms of composition at the order rank in the three subregions p-value &amp;lt; 0.01). These differences were mainly driven by Anoecales, Peridiniales, Borokales, Paraliales and Gonyaulacales, which together contributed almost 80% of the average dissimilarity. Anoecales was the dominant order in the Ionian – Central Mediterranean and Adriatic Sea, but with considerably different relative abundances (52% and 36%, respectively), while Borokales was the dominant order in the Western Mediterranean Sea (33%). Similarly, significant differences among the three marine subregions were also highlighted when protist assemblages were examined in terms of trophic function, both in terms of alpha diversity (calculated on the ASVs for each trophic group) and community composition p-value &amp;lt; 0.01. In particular, the Adriatic Sea stood out for having the highest relative abundance of autotrophic/mixotrophic components in the surface sediments analyzed. Conversely, no significant differences in protist assemblages were found among depth groups. This study provided new insights into the taxonomic and trophic composition of benthic protist communities found in Mediterranean surface sediments, revealing geographical differences among regional seas. The results were discussed in relation to the Mediterranean environmental features that could generate the differences among benthic protist communities.

Co-metabolic growth and microbial diversity: Keys for the depletion of the α, δ, β and γ-HCH isomers

The objective of this study was the isolation and enrichment of microbiomes capable of degrading the main hexachlorocyclohexane isomers quantified in environmental matrices, e.g.: the α, δ, β and γ-HCH isomers. Four microbiomes were isolated and enriched from an HCH-contaminated dumpsite in Italy, both in the presence of HCH isomers (1:1:1:1) as the sole carbon sources and under co-metabolic growth conditions in presence of glucose (0.1 % v/v). The microbiomes were assessed for their relevant metabolic capabilities. A quantitative metabarcoding approach was employed to analyze the compositional evolution of the four microbiomes during the enrichment phase and the phase of testing of the HCH isomers degradation kinetics. The use of a co-metabolic substrate during enrichment process was essential for selecting microbiomes with higher biodiversity. All microbiomes efficiently degraded the α, δ, and γ-HCH isomers. The highest efficiency in the β-HCH degradation capacity was positively correlated to the highest biodiversity of the microbiome, and the involvement of Chryseobacterium and Asinibacterium sps. have been proposed for a recorded increment in bacterial load during the HCH degradation process.

Zoonotic potential of urban wildlife faeces, assessed through metabarcoding

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.

The Diatom Diversity and Ecological Status of a Tufa-Depositing River through eDNA Metabarcoding vs. a Morphological Approach-A Case Study of the Una River (Bosnia and Herzegovina).

Tufa deposits in karst rivers are unique habitats created by mutual interactions between specific environmental and biotope features and inhabited by diatoms as a highly abundant and diverse algal group. This pilot study aimed to investigate the diversity of diatom communities on tufa depositing habitats and assess the Una River's ecological status using a comparative molecular and morphological approach for diatom identification. The 312 base pairs of the rbcL gene were barcoded and analyzed using MiSeq reads and amplicon sequence variants (ASVs) obtained by the DADA2 pipeline. The reference database Diat.barcode v7 was used for taxonomic assignment. The morphological identification of the diatoms was carried out in parallel. In total, the combined dataset revealed 46 taxa identified at genus rank, 125 on the subgenus, and 145 on combined taxonomy rank. The metabarcoding approach mostly leads to a lower number of identified taxa at species rank (58 in molecular vs. 119 in optical inventory), resulting in higher values of beta diversity and heterogeneity in diatom assemblages in samples obtained by morphological approach. Despite the high percentage of taxonomically not assigned diatom ASVs to the species rank, high Shannon diversity index values and a similar number of taxa per locations compared to the morphological approach were obtained. Taxa Achnanthidium minutissimum (Kützing) Czarnecki, Achnanthidium pyrenaicum (Hustedt) H.Kobayasi, Amphora pediculus (Kützing) Grunow, Diatoma vulgaris Bory, Navicula cryptotenella Lange-Bertalot, and Navicula tripunctata (O.F.Müller) Bory were identified at all locations in both inventories. Although limited consistency in the diatom abundances between the two inventory datasets was found, a similar grouping of samples was observed connected to the river's longitudinal gradient. The data obtained using molecular approach in most sites indicated a mostly lower ecological status (good or moderate) compared to the data obtained from the morphological approach (high, good, and moderate). The potential of environmental DNA (eDNA) diatom metabarcoding for water monitoring and diversity studies is undeniable, but to fully realize the benefits of these methods in the future, it is essential to standardize protocols and expand the reference database for species found in specific habitats, such as tufa deposits.