Exploring the impact of urban pollution on ciliate diversity along the Sapucaí River (Minas Gerais, Brazil) via DNA metabarcoding.

BACKGROUND: Protists are diverse single-celled eukaryotes found in various habitats. They exhibit a wide range of forms and functions, representing a significant portion of the eukaryotic tree of life, which also includes animals, plants, and fungi. Due to their high sensitivity to environmental changes, these organisms are widely used as biological indicators of organic pollution. METHODS AND RESULTS: We investigated the molecular diversity of ciliate protists at seven strategic points along the Sapucaí River (Itajubá, Minas Gerais State, Brazil), to assess the impact of urban pollution on the richness, abundance, and diversity indexes of these communities. For each sampling point, values of physicochemical parameters were also recorded. DNA sequences were obtained by high-throughput sequencing (HTS) and analyzed using the V4 18S-rRNA molecular marker, employing the DNA metabarcoding method. We recorded 125 ciliate taxonomic units (OTUs), with nearly 80% corresponding to the classes Spirotrichea, Oligohymenophorea, and Litostomatea. At the genus level, 54 OTUs (43.2%) were identified, spanning 28 genera. CONCLUSIONS: The composition of ciliates varied significantly along the river's course, from upstream to downstream of Itajubá city. Samples collected from the urban area displayed the lowest richness and diversity, corroborating the influence of the pollution gradient on these communities. The physicochemical parameters showed little variation among the samples and were not linked to the observed changes in ciliate communities, revealing that these organisms are strongly affected by environmental changes and respond more sensitively to these disturbances than physicochemical parameters, emphasizing their potential as bioindicators.

Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free.

BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing. CONCLUSION: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

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.

Plastome Evolution and Comparative Analyses of a Recently Radiated Genus Vanda (Aeridinae, Orchidaceae).

Vanda R.Br. is an epiphytic orchid genus with significant horticultural and ornamental value. Previous molecular studies expanded Vanda including some members from five other genera. However, the interspecific relationships of this recently radiated genus have remained unclear based on several DNA markers until now. In this study, the complete plastome has been used to infer the phylogenetic relationships of Vanda s.l. The five newly obtained plastomes ranged from 146,340 bp to 149,273 bp in length, with a GC content ranging from 36.5% to 36.7%. The five plastomes contained 74 protein-coding genes (CDSs), 38 tRNAs, and 8 rRNAs, and their ndh genes underwent loss or pseudogenization. Comparative plastome analyses of 13 Vanda species revealed high conservation in terms of genome size, structure, and gene order, except for a large inversion from trnGGCC to ycf3 in V. coerulea. Moreover, six CDSs and five non-CDSs were selected as candidate DNA barcodes. Our phylogenetic analyses demonstrated that Vanda s.l. is a monophyletic group with high supporting values based on five different datasets (complete plastome with one IR, 68 CDSs, LSC, five hypervariable non-CDSs, and six hypervariable CDSs), while the phylogenetic relationships among species were fully resolved based on the complete plastome with one IR dataset. Our results confirmed that the complete plastome has a great power in resolving the phylogenetic relationships of recently radiated lineages.

Using grass inflorescence as source material for biomonitoring through environmental DNA metabarcoding.

BACKGROUND: Over the last decade, increasing attention has been directed to using different substrates as sources of environmental DNA (eDNA) in ecological research. Reports on the use of environmental DNA located on the surface of plant leaves and flowers have highlighted the utility of this DNA source in studies including, but not limited to, biodiversity, invasive species, and pollination ecology. The current study assesses grass inflorescence as a source of eDNA for detecting invertebrate taxa. METHODS AND RESULTS: Inflorescences from four common grass species in a central South African grassland were collected for high-throughput sequencing analysis. Universal COI primers were utilised to detect Metazoan diversity. The sequencing results allowed for the detection of three Arthropoda orders, with most OTUs assigned to fungal taxa (Ascomycota and Basidiomycota). Some biases were detected while observing the relative read abundance (RRA) results. DISCUSSION: The observed biases could be explained by the accidental inclusion of invertebrate specimens during sample collection and DNA extraction. Primer biases towards the amplified taxa could be another reason for the observed RRA results. This study provided insight into the invertebrate community associated with the four sampled grass species. It should be noted that with the lack of negative field controls, it is impossible to rule out the influence of airborne eDNA on the observed diversity associated with each grass species. The lack of the inclusion of PCR and extraction blanks in the sequencing step, as well as the inclusion of negative field controls, including other areas for refinement were highlighted, and suggestions were provided to improve the outcomes of future studies.

From land to sea: Environmental DNA is correlated with long-term water quality indicators in an urbanized estuary.

Estuaries provide critical ecosystem services, and yet are increasingly under threat from urbanization. Non-invasive approaches to monitor biodiversity resident to or migrating through estuaries is critical to evaluate the holistic health of these ecosystems, often based entirely on water quality. In this study we compared tree of life metabarcoding (ToL-metabarcoding) biodiversity detections with measurements of physico-chemical variables (chlorophyll a, turbidity, total nitrogen, total phosphorous, dissolved oxygen) at eight sites of varying degrees of water quality in the Gold Coast Broadwater Estuary (Queensland, Australia). These sites were ranked according to an adapted Water Quality Index (WQI) score. Here, we detected 787 unique taxa, adding 137 new biodiversity records to the region, mostly micro-organisms such as bacteria, ciliates, diatoms, dinoflagellates, and cryptomonads. Sites with the lowest WQI were characterised by higher turbidity, lower dissolved oxygen, as well as higher total nitrogen and phosphorous, which correlated with an increased diversity of bacteria, ciliates, and green algae. Similarly, the composition of taxa was significantly different between sites with variable WQI values for most taxa but was less apparent for larger vertebrate groups. These findings suggest that rapid ToL-metabarcoding biodiversity detections, particularly for lower order taxonomic groups, can serve as valuable indicators of flora and fauna across the tree of life associated with dynamically shifting estuarine health along urbanized coastlines.

Report of a new species of sand fly, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), from Western Ghats, India.

BACKGROUND: Western Ghats is a biodiversity treasure trove with reports of indigenous leishmaniasis cases. Hence, systematic sand fly surveillance was carried out among the tribal population. The present study reports a novel sand fly species, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), discovered in the Western Ghats of India. METHODS: A comprehensive sand fly survey was conducted across the Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts of Kerala, India. The survey spanned both indoor and outdoor habitats using standard collection methods over a 3-year, 3-month period. DNA barcoding of samples was performed targeting mitochondrial cytochrome c oxidase subunit I (COI) gene, and the sequence generated was subjected to phylogenetic analysis. RESULTS: Phlebotomus (Anaphlebotomus) ajithii, a new sand fly species, is recorded and described in this communication. The morphological relationship of the new species to other members of the subgenus Anaphlebotomus is discussed. Mitochondrial COI barcode followed by phylogenetic analysis confirmed that specimens of Ph. ajithii belong to the same taxonomic group, while a genetic distance of 11.7% from congeners established it as a distinct species. CONCLUSIONS: The Western Ghats, known for its rich biodiversity, has lacked systematic entomological surveys focusing on sand flies. This study aims to fill this gap and reports and describes a new species of sand fly.

Insights into malaria vectors-plant interaction in a dryland ecosystem.

Improved understanding of mosquito-plant feeding interactions can reveal insights into the ecological dynamics of pathogen transmission. In wild malaria vectors Anopheles gambiae s.l. and An. funestus group surveyed in selected dryland ecosystems of Kenya, we found a low level of plant feeding (2.8%) using biochemical cold anthrone test but uncovered 14-fold (41%) higher rate via DNA barcoding targeting the chloroplast rbcL gene. Plasmodium falciparum positivity was associated with either reduced or increased total sugar levels and varied by mosquito species. Gut analysis revealed the mosquitoes to frequently feed on acacia plants (~ 89%) (mainly Vachellia tortilis) in the family Fabaceae. Chemical analysis revealed 1-octen-3-ol (29.9%) as the dominant mosquito attractant, and the sugars glucose, sucrose, fructose, talose and inositol enriched in the vegetative parts, of acacia plants. Nutritional analysis of An. longipalpis C with high plant feeding rates detected fewer sugars (glucose, talose, fructose) compared to acacia plants. These results demonstrate (i) the sensitivity of DNA barcoding to detect plant feeding in malaria vectors, (ii) Plasmodium infection status affects energetic reserves of wild anopheline vectors and (iii) nutrient content and olfactory cues likely represent potent correlates of acacia preferred as a host plant by diverse malaria vectors. The results have relevance in the development of odor-bait control strategies including attractive targeted sugar-baits.

Marine catfishes (Ariidae-Siluriformes) from the Coastal Amazon: mitochondrial DNA barcode for a recent diversification group?

Background: Ariidae species play a significant role as fishing resources in the Amazon region. However, the family's systematic classification is notably challenging, particularly regarding species delimitation within certain genera. This difficulty arises from pronounced morphological similarities among species, posing obstacles to accurate species recognition. Methods: Following morphological identification, mitochondrial markers (COI and Cytb) were employed to assess the diversity of Ariidae species in the Amazon. Results: Our sampling efforts yielded 12 species, representing 92% of the coastal Amazon region's diversity. Morphological identification findings were largely corroborated by molecular data, particularly for species within the Sciades and Bagre genera. Nonetheless, despite morphological support, Cathorops agassizii and Cathorops spixii displayed minimal genetic divergence (0.010). Similarly, Notarius quadriscutis and Notarius phrygiatus formed a single clade with no genetic divergence, indicating mitochondrial introgression. For the majority of taxa examined, both COI and Cytb demonstrated efficacy as DNA barcodes, with Cytb exhibiting greater polymorphism and resolution. Consequently, the molecular tools utilized proved highly effective for species discrimination and identification.

Mismanagement and poor transparency in the European processed seafood supply revealed by DNA metabarcoding.

In the global processed seafood industry, disparate actors play different roles along the supply chain, creating multiple opportunities for mistakes, malpractice, and fraud. As a consequence, consumers may be exposed to non-authentic products, which hinder informed purchasing decisions and broader efforts to improve trade transparency and sustainability. Here, we characterised the taxonomic composition of 62 processed seafood products in Italian, British and Albanian retailers, purposefully obtained from different supply routes, using multiple DNA metabarcoding markers. By combining molecular results with metadata reported on labels, we revealed patterns of mislabelling in 24 products (39%) across sampling regions, denoting lack of transparency of processed seafood products based on resources sourced from either Europe or globally. We show that the accuracy of label claims and the mis-represented and underestimated levels of traded biodiversity are largely determined by the management of raw material by global processors. Our study shows that DNA metabarcoding is a powerful and novel authentication tool that is mature for application at different stages of the seafood supply chain to protect consumers and improve the sustainable management of fish stocks.

Unveiling ophiuroid biodiversity across North Atlantic habitats via an integrative perspective.

The depths of the North Atlantic Ocean host a species-rich fauna providing heterogeneous habitats from thermal vent fields to cold-water coral reefs. With the increasing threat of destruction of deep-sea habitats due to human impacts, such as demersal fishing and the beginning of deep-sea mining, an analysis of the diversity and distribution of species is crucial for conservation efforts. Brittle stars occur in high biomasses, contributing to the biodiversity of the seafloor. Specimens were collected during several scientific expeditions to gain a more detailed insight into the brittle star diversity in the North Atlantic Ocean. An integrative approach to identify the species with DNA barcoding (mtCOI) in combination with morphological studies revealed 24 species. Most species have been previously identified in the North Atlantic, but sequences for 13 species are newly added to public repositories. Additionally, the MALDI-TOF-MS proteomic analysis was successfully applied for 197 specimens with known COI barcodes. Results are congruent with other molecular species delimitations demonstrating the functionality of proteomics for the identification of brittle stars. This dataset significantly expands our understanding of the taxonomic and genetic diversity of brittle stars and contributes to publicly available data. It emphasizes the importance of considering habitat heterogeneity for large scale patterns of biodiversity.

Determining ecological interactions of key dinoflagellate species using an intensive metabarcoding approach in a semi-closed coastal ecosystem of South Korea.

Marine phytoplankton communities are pivotal in biogeochemical cycles and impact global climate change. However, the dynamics of the dinoflagellate community, its co-occurrence relationship with other eukaryotic plankton communities, and environmental factors remain poorly understood. In this study, we aimed to analyze the temporal changes in the eukaryotic plankton community using a 18S rDNA metabarcoding approach. We performed intensive monitoring for 439 days at intervals of three days during the period from November 2018 to June 2020 (n = 260) in Jangmok Bay Time-series Monitoring Site in South Korea. Among the 16,224 amplicon sequence variants (ASVs) obtained, dinoflagellates were the most abundant in the plankton community (38 % of total relative abundance). The dinoflagellate community was divided into 21 groups via cluster analysis, which showed an annually similar distribution of low-temperature periods. Additionally, we selected 11 taxa that had an occurrence mean exceeding 1 % of the total dinoflagellate abundance, accounting for 93 % of the total dinoflagellate community: namely Heterocapsa rotundata, Gymnodinium sp., Akashiwo sanguinea, Amoebophrya sp., Euduboscquella sp., Spiniferites ramosus, Dissodinium pseudolunula, Sinophysis sp., Karlodinium veneficum, and Katodinium glaucum. The key dinoflagellate species were well represented at temporally variable levels over an entire year. Heterocapsa rotundata was not significantly affected by water temperature, whereas its dynamics were largely influenced by strong predation pressure, competition, and/or the supplementation of food sources. The growth of A. sanguinea was associated with dissolved inorganic phosphorus concentrations, while Euduboscquella sp. showed a significant relationship with D. pseudolunula and K. glaucum, largely representing a positive association that implies possible parasitic mechanisms. This study demonstrated interactions between key dinoflagellate species and the environment, as well as parasites, predators, competitors, and feeders.

Cost-efficient PCR based DNA barcoding of marine invertebrate specimens with NovaSeq amplicon sequencing.

BACKGROUND: The marine environment harbors high biodiversity; however, it is poorly understood. Nucleotide sequence data of all marine organisms should be accumulated before natural and/or anthropogenic environmental changes jeopardize the marine environment. In this study, we report a cost-effective and easy DNA barcoding method. This method can be readily adopted without using library preparation kits. It includes multiplex PCR of short targets, indexing PCR, and outsourcing to a sequencing service using the NovaSeq system. METHODS AND RESULTS: We targeted four mitochondrial genes [cytochrome c oxidase subunit I (COI), COIII, 16S rRNA (16S), and 12S rRNA (12S)] and three nuclear genes [18S rRNA (18S), 28S rRNA (28S), internal transcribed spacer 2 (ITS2)] in 95 marine invertebrate specimens, which were primarily annelids. The primers, including adapters and indices for NovaSeq sequencing, were newly designed. Two PCR runs were conducted. The 1st PCR amplified specific loci with universal primers and the 2nd added sequencing adapters and indices to the 1st PCR products. The gene sequences obtained from the FASTQ files were subjected to BLAST search and phylogenetic analyses. One run using 95 specimens yielded sequences averaging 2816 bp per specimen for a total length of six loci. Nuclear genes were more successfully assembled compared with mitochondrial genes. A weak but significantly negative correlation was observed between the average length of each locus and success rate of the assembly. Some of the sequences were almost identical to the sequences obtained from specimens collected far from Japan, indicating the presence of potentially invasive species identified for the first time. CONCLUSIONS: We obtained gene sequences efficiently using next-generation sequencing rather than Sanger sequencing. Although this method requires further optimization to increase the success rate for some loci, it is used as a first step to select specimens for further analyses by determining the specific loci of the targets.

Assessing benthic macroinvertebrate communities' spatial heterogeneity in Mediterranean transitional waters through eDNA metabarcoding.

Transitional waters are important habitats both for biodiversity and ecological functions, providing valuable natural resources and relevant ecosystem services. However, they are highly susceptible to climate changes and anthropogenic pressures responsible for biodiversity losses and require specific biomonitoring programs. Benthic macroinvertebrates are suitable as ecological indicators of transitional waters, being affected by biological, chemical, and physical conditions of the ecosystems about their life cycles and space-use behaviour. The advent of high-throughput sequencing technologies has allowed biodiversity investigations, at the molecular level, in multiple ecosystems and for different ecological guilds. Benthic macroinvertebrate communities' composition has been investigated, at the molecular level, mainly through DNA extracted from sediments in marine and riverine ecosystems. In this work, benthic macroinvertebrate communities are explored through eDNA metabarcoding from water samples in a Mediterranean transitional water ecosystem. This research highlighted the validity of eDNA metabarcoding as an efficient tool for the assessment of benthic macroinvertebrate community structure in transitional waters, unveiling the spatial heterogeneity of benthic macroinvertebrate communities correlated to the measured environmental gradients. The results suggest that peculiar features of transitional water ecosystems, such as shallow waters and limited currents, facilitate the assessment of benthic macroinvertebrate communities through environmental DNA analysis from surface water samples, opening for more rapid and accurate monitoring programs for these valuable ecosystems.

Unravelling large-scale patterns and drivers of biodiversity in dry rivers.

More than half of the world's rivers dry up periodically, but our understanding of the biological communities in dry riverbeds remains limited. Specifically, the roles of dispersal, environmental filtering and biotic interactions in driving biodiversity in dry rivers are poorly understood. Here, we conduct a large-scale coordinated survey of patterns and drivers of biodiversity in dry riverbeds. We focus on eight major taxa, including microorganisms, invertebrates and plants: Algae, Archaea, Bacteria, Fungi, Protozoa, Arthropods, Nematodes and Streptophyta. We use environmental DNA metabarcoding to assess biodiversity in dry sediments collected over a 1-year period from 84 non-perennial rivers across 19 countries on four continents. Both direct factors, such as nutrient and carbon availability, and indirect factors such as climate influence the local biodiversity of most taxa. Limited resource availability and prolonged dry phases favor oligotrophic microbial taxa. Co-variation among taxa, particularly Bacteria, Fungi, Algae and Protozoa, explain more spatial variation in community composition than dispersal or environmental gradients. This finding suggests that biotic interactions or unmeasured ecological and evolutionary factors may strongly influence communities during dry phases, altering biodiversity responses to global changes.

Comprehensive DNA metabarcoding-based detection of non-indigenous invertebrates in recreational marinas through a multi-substrate approach.

eDNA metabarcoding has been increasingly employed in the monitoring of marine invertebrate non-indigenous species (NIS), in particular using filtered seawater. However, comprehensive detection of all NIS may require a diversity of sampling substrates. To assess the effectiveness of 5 sample types (hard and artificial substrates, water, zooplankton) on the recovery of invertebrates' diversity, two marinas were monitored over three time points, using COI and 18S rRNA genes as DNA metabarcoding markers. We detected a total of 628 species and 23 NIS, with only up to 9% species and 17% of NIS detected by all sample types. Hard and artificial substrates were similar to each other but displayed the most significant difference in invertebrate recovery when compared to water eDNA and zooplankton. Five NIS are potential first records for Portugal. No NIS were detected in all sample types and seasons, highlighting the need for varied sampling approaches, and consideration of temporal variation for comprehensive marine NIS surveillance.

The genus Coelastrella (Chlorophyceae, Chlorophyta): molecular species delimitation, biotechnological potential, and description of a new species Coelastrella affinis sp. nov., based on an integrative taxonomic approach.

Despite the long research history on the genus Coelastrella, its species diversity and biotechnological potential have not been fully explored. For the first time, cluster analysis of morphological characteristics was done in the representatives of the said genus. The results obtained have shown that morphological similarity does not necessarily indicate a molecular genetic relationship. It the light of it, the taxonomic status of species can reliably be determined using specific DNA region, such as 18S-ITS1-5.8S-ITS2. The V4 and V9 regions of gene 18S rRNA are relatively conservative fragments which are not suitable for species identification. The ITS2 can be used as a "short barcode". Among the advanced machine methods for delimitation species, the most effective algorithm for distinguishing Coelastrella species was the Generalized Mixed Yule Coalescent (GMYC) method. This paper represented for the first time our comprehensive review of the works devoted to the analysis of the biotechnological potential of representatives of the genus Coelastrella and shows that fatty acid composition of the three main chemogroups within the studied genus differs. In the future, this may form the basis for predicting the composition of the fatty acid profile of new strains, which is important while searching for organisms with specified biotechnological properties. In conclusion, an integrative approach was employed to describe Coelastrella affinis sp. nov., a new species of the genus Coelastrella with high biotechnological potential. Also, a new description of C. thermophila var. astaxanthina comb. nov. was proposed.

Simulation of CRISPR-Cas9 editing on evolving barcode and accuracy of lineage tracing.

We designed a simulation program that mimics the CRISPR-Cas9 editing on evolving barcode and double strand break repair procedure along with cell divisions. Emerging barcode mutations tend to build upon previously existing mutations, occurring sequentially with each generation. This process results in a unique mutation profile in each cell. We sample the barcodes in leaf cells and reconstruct the lineage, comparing it to the original lineage tree to test algorithm accuracy under different parameter settings. Our computational simulations validate the reasonable assumptions deduced from experimental observations, emphasizing that factors such as sampling size, barcode length, multiple barcodes, indel probabilities, and Cas9 activity are critical for accurate and successful lineage tracing. Among the many factors we found that sampling size and indel probabilities are two major ones that affect lineage tracing accuracy. Large segment deletions in early generations could greatly impact lineage accuracy. These simulation results offer insightful recommendations for enhancing the design and analysis of Cas9-mediated molecular barcodes in actual experiments.

North Atlantic deep-sea benthic biodiversity unveiled through sponge natural sampler DNA.

The deep-sea remains the biggest challenge to biodiversity exploration, and anthropogenic disturbances extend well into this realm, calling for urgent management strategies. One of the most diverse, productive, and vulnerable ecosystems in the deep sea are sponge grounds. Currently, environmental DNA (eDNA) metabarcoding is revolutionising the field of biodiversity monitoring, yet complex deep-sea benthic ecosystems remain challenging to assess even with these novel technologies. Here, we evaluate the effectiveness of whole-community metabarcoding to characterise metazoan diversity in sponge grounds across the North Atlantic by leveraging the natural eDNA sampling properties of deep-sea sponges themselves. We sampled 97 sponge tissues from four species across four North-Atlantic biogeographic regions in the deep sea and screened them using the universal COI barcode region. We recovered unprecedented levels of taxonomic diversity per unit effort, especially across the phyla Chordata, Cnidaria, Echinodermata and Porifera, with at least 406 metazoan species found in our study area. These assemblages identify strong spatial patterns in relation to both latitude and depth, and detect emblematic species currently employed as indicators for these vulnerable habitats. The remarkable performance of this approach in different species of sponges, in different biogeographic regions and across the whole animal kingdom, illustrates the vast potential of natural samplers as high-resolution biomonitoring solutions for highly diverse and vulnerable deep-sea ecosystems.

A bud's life: Metabarcoding analysis to characterise hazelnut big buds microbiome biodiversity.

Despite Corylus avellana L. being an economically important shrub species known for its resilience to adverse environmental conditions, it constantly faces attacks from a plethora of biotic entities. Among these, the mite pest Phytoptus avellanae is gaining importance, causing economic losses every year. This mite colonises the new generative and vegetative buds, leading them to become swollen and reddish, and drastically reducing hazelnut production. The biology behind gall formation is still poorly understood. This study provides a qualitative and quantitative description of the microbiome in both healthy and infested buds of two economically important hazelnut cultivars through metabarcoding of fungal ITS and bacterial 16 S. Potentially pathogenic genera such as Fusarium and Pseudomonas were predominant in the infested buds, along with the obligate intracellular bacterial genus Wolbachia. Akanthomyces muscarius was instead isolated from culture-based methods only from the infested buds. These findings could improve the understanding of gall ecology, supporting the management of mite populations, and they could also serve as a milestone for further studies on low-impact, monitoring-driven, and genetically targeted control strategies.