Metabarcoding Analysis Research Articles

Assessing the relevance of DNA metabarcoding compared to morphological identification for lake phytoplankton monitoring

Phytoplankton is a key biological group used to assess the ecological status of lakes. The classical monitoring approach relies on microscopic identification and counting of phytoplankton species, which is time-consuming and requires high taxonomic expertise. High-throughput sequencing, combined with metabarcoding, has recently demonstrated its potential as an alternative approach for plankton surveys. Several studies have confirmed the relevance of the diatom metabarcoding approach to calculate biotic indices based on species ecology. However, phytoplankton communities have not yet benefited from such validation. Here, by comparing the results obtained with the two methods (molecular and microscopic counting), we evaluated the relevance of metabarcoding approach for phytoplankton monitoring by considering different metrics: alpha diversity, taxonomic composition, community structure and a phytoplankton biotic index used to assess the trophic level of lakes. For this purpose, 55 samples were collected in four large alpine lakes (Aiguebelette, Annecy, Bourget, Geneva) during the year 2021. For each sample, a metabarcoding analysis based on two genetic markers (16S and 23S rRNA) was performed, in addition to the microscopic count. Regarding the trophic level of lakes, significant differences were found between index values obtained with the two approaches. The main hypothesis to explain these differences comes from the incompleteness, particularly at the species level, of the barcode reference library for the two genetic markers. It is therefore necessary to complete reference libraries for using such species-based biotic indices with metabarcoding data. Besides this, species richness and diversity were higher in the molecular inventories than in the microscopic ones. Moreover, despite differences in taxonomic composition of the floristic lists obtained by the two approaches, their community structures were similar. These results support the possibility of using metabarcoding for phytoplankton monitoring but in a different way. We suggest exploring alternative approaches to index development, such as a taxonomy-free approach.

Rich diversity and active spatial-temporal dynamics of Thalassiosira species revealed by time-series metabarcoding analysis.

Thalassiosira is a species-rich genus in Bacillariophyta that not only contributes positively as primary producer, but also poses negative impacts on ecosystems by causing harmful algal blooms. Although taxonomical studies have identified a large number of Thalassiosira species, however, the composition of Thalassiosira species and their geographical distribution in marine ecosystems were not well understood due primarily to the lack of resolution of morphology-based approaches used previously in ecological expeditions. In this study, we systematically analyzed the composition and spatial-temporal dynamic distributions of Thalassiosira in the model marine ecosystem Jiaozhou Bay by applying metabarcoding analysis. Through analyzing samples collected monthly from 12 sampling sites, 14 Thalassiosira species were identified, including five species that were not previously reported in Jiaozhou Bay, demonstrating the resolution and effectiveness of metabarcoding analysis in ecological research. Many Thalassiosira species showed prominent temporal preferences in Jiaozhou Bay, with some displaying spring-winter preference represented by Thalassiosira tenera, while others displaying summer-autumn preference represented by Thalassiosira lundiana and Thalassiosira minuscula, indicating that the temperature is an important driving factor in the temporal dynamics. The application of metabarcoding analysis, equipped with appropriate molecular markers with high resolution and high specificity and databases of reference molecular marker sequences for potential all Thalassiosira species, will revolutionize ecological research of Thalassiosira species in Jiaozhou Bay and other marine ecosystems.

Taxonomic resolution of different 16S rRNA variable regions varies strongly across plant-associated bacteria.

Plant-microbiome research plays a pivotal role in understanding the relationships between plants and their associated microbial communities, with implications for agriculture and ecosystem dynamics. Metabarcoding analysis on variable regions of the 16S ribosomal RNA (rRNA) gene remains the dominant technology to study microbiome diversity in this field. However, the choice of the targeted variable region might affect the outcome of the microbiome studies. In our in silico analysis, we have evaluated whether the targeted variable region has an impact on taxonomic resolution in 16 plant-related microbial genera. Through a comparison of 16S rRNA gene variable regions with whole-genome data, our findings suggest that the V1-V3 region is generally a more suitable option than the widely used V3-V4 region for targeting microbiome analysis in plant-related genera. However, sole reliance on one region could introduce detection biases for specific genera. Thus, we are suggesting that while transitioning to full-length 16S rRNA gene and whole-genome sequencing for plant-microbiome analysis, the usage of genus-specific variable regions can achieve more precise taxonomic assignments. More broadly, our approach provides a blueprint to identify the most discriminating variable regions of the 16S rRNA gene for genera of interest.

The high molecular diversity in Noctiluca scintillans is dominated by intra-genomic variations revealed by single cell high-throughput sequencing of 18S rDNA V4

The application of high-throughput sequencing (HTS) technologies has revolutionized research on phytoplankton biodiversity by generating an unprecedented amount of molecular data in marine ecosystem surveys. However, high-level of molecular diversity uncovered in HTS-based metabarcoding analyses may lead to overinterpretation of phytoplankton diversity due to excessive intra-genomic variations (IGVs). The aims in this study are to explore the nature of phytoplankton molecular diversity and to test the hypothesis. We carried out single-cell metabarcoding analysis of 18S rDNA V4 sequences obtained in single Noctiluca scintillans cells isolated from various sites in coastal waters of China. Results showed that each single N. scintillans cell harbored a high level of IGVs with about 100 amplicon sequence variants (ASVs). The large numbers of non-dominant ASVs identified in N. scintillans cells, which might correspond to the larger numbers of ASVs annotated as N. scintillans and showed similar temporal dynamics in metabarcoding analyses, could inflate the inter-species diversity or intra-species genetic diversity. In addition, there were large numbers of additional ASVs that were not annotated as N. scintillans. These non-N. scintillans ASVs might represent diverse preys for N. scintillans, consistent with previous reports that N. scintillans may act as chance predator of a broad-spectrum preys. This single-cell study has unambiguously demonstrated that the existence of high levels of IGVs in N. scintillans and most likely many other phytoplankton species, demonstrating that the majority of the molecular diversity revealed in metabarcoding analysis, which were generally interpreted as the sum of inter-species diversity and intra-species diversity, actually included high levels of IGVs and should be interpreted with caution.

Forest management and former land use have no effect on soil fungal diversity in uneven-aged mountain high forests

Key messageMetabarcoding analysis of soil fungal communities in French mountain forests revealed that harvesting intensity, time since last harvest and former land use had no effect on fungal community composition compared to key abiotic factors. Low-intensity management in these uneven-aged mountain forests therefore has limited effects on soil fungal community composition which is mainly driven by elevation and edaphic properties.ContextPast and current human activities are known to affect forest biodiversity. However, the effects of former land use and forest management have been studied much more extensively on higher plants than on fungi.AimsOur objectives were to assess the effects of harvesting intensity, duration since last harvest and former land use on soil fungal communities in uneven-aged mountain high forests.MethodsOn the basis of historical land-use maps drawn between 1862 and 1864 and on historical forest management archives, we selected 62 sites in the French Alps with contrasting land-use histories (ancient forests, which were already forested on historical maps vs recent forests, which have recovered following abandonment of pastures) and different durations since last harvest (from 1 to over 50 years). We carried out soil sampling and assessed fungal diversity by metabarcoding analysis. We analysed soil fungal molecular operational taxonomic units (MOTU) diversity as a whole and for the main lifestyle groups (such as wood saprotrophic or ectomycorrhizal fungi) using multiple linear regressions on Shannon’s diversity index and fungal taxonomic composition using canonical correlation analysis.ResultsWe found no significant effect of harvesting intensity, time since last harvest or land-use history on total fungal MOTU diversity, fungal lifestyle diversity or taxonomic composition. In contrast, we observed significant effects of elevation, pH, organic carbon and available phosphorus content on the taxonomic and functional composition of soil fungal communities.ConclusionsThe structure of soil fungal communities (i.e. diversity and species composition) was mainly determined by elevation and edaphic factors, indicating a high-context dependency, as previously found in similar studies. Our study in mountain forests shows that recent forests established on former pastures had no legacy effect on soil conditions and fungal communities, in contrast to previous results in lowland areas, where recent forests were mainly established on former cropland. Uneven-aged forest management had no effect on fungal diversity, in contrast to previous results observed in even-aged high forests.

Risk Screening of Invasive Aquatic Species and a Survey of Fish Diversity Using Environmental DNA Metabarcoding Analysis in Shanghai

As the largest coastal city in China, Shanghai’s rapid development in transportation, tourism, trade, and commerce has facilitated the spread and invasion of non-native aquatic organisms. Aquatic organisms are highly elusive, and once established, eradicating them becomes a challenging task. Currently, our understanding of the invasion risk posed by non-native aquatic species in Shanghai is limited. Therefore, it is imperative to investigate the pathways of introduction, distribution, and dispersion and the invasion risk and impacts of non-native aquatic organisms in Shanghai. This study investigated aquatic organisms in Shanghai’s primary water bodies, including Huangpu River, Suzhou River, and Dianshan Lake. The risk assessment was conducted using the Aquatic Species Invasiveness Screening Kit (AS-ISK), and field monitoring was performed with environmental DNA (eDNA) technology. Results of the risk assessment indicate that among the 21 evaluated species, 9 fall into the medium-to-high-risk category with scores ≥26, while 12 are classified as low-risk with scores <26. The top four species with the highest invasion risk are Gambusia affinis, Pomacea canaliculata, Lepomis macrochirus, and Coptodon zillii. This study identified 54 fish species belonging to seven orders, 16 families, and 42 genera at 16 sampling sites in Shanghai, among which Channa maculata, Micropterus salmoides, and Misgurnus bipartitus are non-native. The results suggest that Shanghai faces a high invasion risk of aquatic species, necessitating enhanced scientific prevention and control measures. Early monitoring is essential for species with medium-to-high invasion risk, and a further evaluation and analysis of the risks associated with introduced fish species already present in Shanghai are recommended for aquaculture practices.

Perspectives from an eDNA metabarcoding pilot project in the Banggai MPA, Central Sulawesi, Indonesia

The Banggai MPA is one of many Indonesian MPAs established or reestablished as a result of the shift from regency to provincial level governance over waters 0–4 NM from shore under Act 23/2014. The MPA covers a substantial proportion of Banggai Archipelago coastal ecosystems and around 90% of endangered Banggai cardinalfish (Pterapogon kauderni) endemic habitat. An expedition in 1998 reported high fish biodiversity; however, logistics and taxonomic expertise requirements mean that similar methods are not realistic for collecting baseline or monitoring biodiversity data to support MPA management. A pilot project in 2018 collected water samples (four shallow-water coastal sites, three replicates per site) around Banggai Island for environmental DNA (eDNA) metabarcoding using two molecular markers: COI mtDNA and 12S rRNA. COI data were overwhelmingly dominated by a few phytoplankton species, reflecting challenges using this marker in areas with dense phytoplankton, and could not be analyzed further. The 12S rRNA sequences were processed and aggregated into amplicon sequence variants (ASVs) using the Anacapa Toolkit. These data provide a snapshot of fish diversity by site, although accumulation curves indicate three samples per site is insufficient to provide a comprehensive record. Identified taxa overlapped and exceeded in number those detected in 1998. The percentage of ASVs unassigned at species and genus level (e.g., gobies and cardinalfishes) and taxonomic ambiguities indicate a need for combined classical taxonomic and barcoding studies to enhance nucleotide databases (GenBank/BOLD), ideally with regional voucher specimen repositories. Furthermore, the study revealed the need for further capacity building in metabarcoding data analysis.

Exploring freshwater snail diversity and community structure in china's largest lake using eDNA technology

Freshwater snails are a crucial benthic animal group that plays a pivotal role in algae regulation and ecosystems while serving as intermediate hosts for certain parasites. Environmental DNA (eDNA) technology offers numerous advantages for biodiversity assessment. However, the utilization of eDNA techniques for freshwater snail monitoring remains limited. In this study, we screened primers for assessing freshwater snail diversity using eDNA technology and constructed a comprehensive database for Poyang Lake. Using eDNA technology, a total of 21 species of snails were detected in Poyang Lake, while traditional methods detected only 11 species. Notably, all species collected by traditional methods were also detected by eDNA technology. Additionally, environmental DNA methods detected the invasive snail Pomacea canaliculata, while traditional methods did not. In this study, eleven intermediate host snail species for parasitic disease were identified using eDNA methods, while six were detected through traditional sampling techniques. The eDNA metabarcoding analysis showed that the α and β diversity levels of snails in Poyang Lake did not vary significantly, indicating that results obtained by eDNA sampling are not influenced by the season in which samples were collected. The correlation analysis between the community structure of snails and environmental factors in Poyang Lake revealed significant impacts of total nitrogen, chlorophyll a, water depth, and water temperature on the snail community structure. These results confirm that eDNA technology can be exploited to monitor freshwater snail biodiversity.

Microbial composition of Saharan dust plumes deposited as red rain in Granada (Southern Spain)

During durst storms, also biological material is transported from arid areas such as the Sahara Desert. In the present work, rain samples containing significant amounts of mineral dust have been collected in Granada during different red rain episodes. Biological features (bacteria, biofilm, pollen grain and fungal spore) as well as size-particle distribution and mineralogical composition were studied by SEM. Nanobacteria were observed for the first time in red rain samples. A preliminary metabarcoding analysis was performed on three red rain samples. Here, Bacillota made up 18 % and Pseudomonadota 23 % of the whole prokaryotic community. The fungal community was characterized by a high abundance of Ascomycota and, dependent on the origin, the presence of Chytridiomycota. By means of 16S rRNA sequencing, 18 cultivable microorganisms were identified. In general, members of the phyla Pseudomonadota and Bacillota made up the majority of taxa. Some species, such as Peribacillus frigoritolerans and Bacillus halotolerans were isolated during three different red rain episodes. Generally, red rain carries a wide variety of microorganisms, being their ecosystem and health effects largely unknown.

Metabarcoding analysis reveals a differential bacterial community profile associated with ‘Torta del Casar’ and ‘Queso de la Serena’ PDO cheeses

A detailed understanding of the microbial ecology of artisanal cheeses is a crucial factor for implementation practices that can help to update their quality. In this study, the bacterial communities of Protected Designation of Origin (PDO) ‘Queso de la Serena’ and ‘Torta del Casar’ cheeses were characterised by 16S rRNA gene metabarcoding sequencing over 60 days of ripening at three dairies for each cheese variety. Both PDO cheeses presented a distinctive and unique bacterial community, suggesting that the manufacturing practices of each PDO shaped the microbiota. Lactococcus lactis and, to a lesser extent, Leuconostoc mesenteroides were the key lactic acid bacteria (LAB) species that drove the ripening process of PDO ‘Torta del Casar’, whereas in PDO ‘Queso de la Serena’, Leu. mesenteroides was the main LAB species, followed by Lactococcus raffinolactis and Lc. lactis, depending on the dairy. Furthermore, a wide range of non-starter LAB, Gram-negative bacteria and Gram-positive catalase-positive cocci were found, with a variable presence and abundance between dairies. In this regard, the dairy was a strong contributing factor to the variability in the composition of the bacterial community in each cheese type. In contrast, the impact of the ripening process was limited. Overall, this study enhances the understanding of the bacterial dynamics that occur during the ripening of these traditional PDO cheeses, highlighting the need to improve the standardisation of the raw materials and manufacturing practices to minimise inter-dairy variability, ensuring final quality and their unique sensory traits.

Covariation between microeukaryotes and bacteria associated with Planorbidae snails.

Microbial communities associated with macroorganisms might affect host physiology and homeostasis. Bacteria are well studied in this context, but the diversity of microeukaryotes, as well as covariations with bacterial communities, remains almost unknown. To study microeukaryotic communities associated with Planorbidae snails, we developed a blocking primer to reduce amplification of host DNA during metabarcoding analyses. Analyses of alpha and beta diversities were computed to describe microeukaryotes and bacteria using metabarcoding of 18S and 16S rRNA genes, respectively. Only three phyla (Amoebozoa, Opisthokonta and Alveolata) were dominant for microeukaryotes. Bacteria were more diverse with five dominant phyla (Proteobacteria, Bacteroidetes, Tenericutes, Planctomycetes and Actinobacteria). The composition of microeukaryotes and bacteria were correlated for the Biomphalaria glabrata species, but not for Planorbarius metidjensis. Network analysis highlighted clusters of covarying taxa. Among them, several links might reflect top-down control of bacterial populations by microeukaryotes, but also possible competition between microeukaryotes having opposite distributions (Lobosa and Ichthyosporea). The role of these taxa remains unknown, but we believe that the blocking primer developed herein offers new possibilities to study the hidden diversity of microeukaryotes within snail microbiota, and to shed light on their underestimated interactions with bacteria and hosts.

MiscMetabar: an R package to facilitate visualization and reproducibility in metabarcoding analysis

MiscMetabar: an R package to facilitate visualization and reproducibility in metabarcoding analysis

Bat diversity boosts ecosystem services: Evidence from pine processionary moth predation

Coniferous forests contribute to the European economy; however, they have experienced a decline since the late 1990s due to an invasive pest known as the pine processionary moth, Thaumetopoea pityocampa. The impacts of this pest are increasingly exacerbated by climate change. Traditional control strategies involving pesticides have had negative effects on public health and the environment. Instead, forest managers seek a more ecological and sustainable approach to management that promotes the natural actions of pest control agents. This study aims to evaluate the role of bats in suppressing pine processionary moths in pine forests and examine how the bat community composition and abundance influence pest consumption. Bats were sampled in the mountainous environment of the Serra da Estrela in central Portugal to collect faecal samples for DNA meta-barcoding analysis. We assessed the relationship between a) bat richness, b) bat relative abundance, c) bat diet richness, and the frequency of pine processionary moth consumption. Our findings indicate that sites with the highest bat species richness and abundance exhibit the highest levels of pine processionary moth consumption. The intensity of pine processionary moth consumption is independent of insect diversity within the site. The highest occurrence of pine processionary moth presence in bat diets is primarily observed in species that forage in cluttered habitats. A typical predator of pine processionary moths among bats is likely to be a forest-dwelling species that specialises in consuming Lepidoptera. These species primarily use short-range echolocation calls, which are relatively inaudible to tympanate moths, suitable for locating prey in cluttered environments, employing a gleaning hunting strategy. Examples include species from the genera Plecotus, Myotis, and Rhinolophus. This study enhances our understanding of the potential pest consumption services provided by bats in pine forests. The insights gained from this research can inform integrated pest management practices in forestry.

Protists in hypoxic waters of Jinhae Bay and Masan Bay, Korea, based on metabarcoding analyses: emphasizing surviving dinoflagellates

Hypoxia can indeed impact the survival of protists, which play a crucial role in marine ecosystems. To better understand the protistan community structure and species that can thrive in hypoxic waters, we collected samples from both the surface and bottom waters during the hypoxic period in Jinhae and Masan Bays and the non-hypoxic period in Jinhae Bay. Subsequently, we utilized metabarcoding techniques to identify the protistan species. During hypoxia, with dissolved oxygen concentrations of 0.8 mg L<sup>-1</sup> in Jinhae Bay and 1.8 mg L<sup>-1</sup> in Masan Bay within the bottom waters, the phylum Dinoflagellata exhibited the highest amplicon sequence variants richness among the identified protist phyla. Following the Dinoflagellata, Ochrophyta and Ciliophora also displayed notable presence. In hypoxic waters of Jinhae and Masan Bays, we identified a total of 36 dinoflagellate species that exhibited various trophic modes. These included one autotrophic species, 14 mixotrophic species, 9 phototrophic species with undetermined trophic modes (either autotrophic or mixotrophic), 2 kleptoplastidic species, and 10 heterotrophic species. Furthermore, the hypoxic bottom water exhibited a greater number of heterotrophic dinoflagellate species compared to the non-hypoxic surface water within the same water column or the non-hypoxic bottom water. Therefore, feeding by mixotrophic and heterotrophic dinoflagellates may be partially responsible for their dominance in terms of the number of species surviving in hypoxic waters. This study not only introduces the initial documentation of 26 dinoflagellate species surviving in hypoxic conditions but also establishes a foundation for a more comprehensive understanding of the ecophysiology of dinoflagellates in hypoxic marine environments.

Microbial communities of a variety of 75 homemade fermented vegetables.

Fermentation is an ancient practice of food preservation. Fermented vegetables are popular in Eastern European and Asian countries. They have received a growing interest in Western countries, where they are mainly manufactured at domestic and artisanal scales and poorly characterized. Our aim was to investigate the microbial communities and the safety of French homemade fermented vegetables, in the frame of a citizen science project. Fermented vegetables and the data associated with their manufacture were collected from citizens and characterized for pH, NaCl concentration, and microbiology by culturomics and 16S DNA metabarcoding analysis. Lactic acid bacteria (LAB) and yeast isolates were identified by 16S rRNA gene sequencing and D1/D2 domains of the large subunit of the rRNA gene, respectively. The 75 collected samples contained 23 types of vegetables, mainly cabbage, followed by carrots and beets, and many mixtures of vegetables. They were 2 weeks to 4 years old, and their median pH was 3.56, except for two samples with a pH over 4.5. LAB represented the dominant viable bacteria. LAB concentrations ranged from non-detectable values to 8.7 log colony-forming units (CFU)/g and only depended on the age of the samples, with the highest most frequently observed in the youngest samples (<100 days). The 93 LAB isolates identified belonged to 23 species, the two mains being Lactiplantibacillus pentosus/plantarum and Levilactobacillus brevis. The other microbial groups enumerated (total aerobic bacteria, halotolerant bacteria, Gram-negative bacteria, and acetic acid bacteria) generally showed lower concentrations compared to LAB concentrations. No pathogenic bacteria were detected. Viable yeasts were observed in nearly half the samples, at concentrations reaching up to 8.0 log CFU/g. The 33 yeast clones identified belonged to 16 species. Bacterial metabarcoding showed two main orders, namely, Lactobacillales (i.e., LAB, 79% of abundance, 177 of the 398 total ASVs) and Enterobacterales (19% of abundance, 191 ASVs). Fifteen LAB genera were identified, with Lactiplantibacillus and Levilactobacillus as the most abundant, with 41 and 12% of total reads, respectively. Enterobacterales members were mainly represented by Enterobacteriaceae and Yersiniaceae. This study is the first wide description of the microbiota of a large variety of homemade fermented vegetables and documents their safety.

Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses

The research presented investigates whether DNA-based metabarcoding can replace the morphology-based identification of diatom taxa in the ecological status assessments of aquatic habitats. When comparing data obtained with microscopy and metabarcoding, significant deviations have been noticed. One of the main reasons includes the incompleteness of the reference database used for taxonomic annotation of sequences. The database library should be complemented with species inhabiting unique habitats and having specific environmental requirements representing environmental endpoints for genetic diversification. Soda pans and soda lakes are examples of an extreme habitat with the loss of sodic character as the main threat; thus, accurate identification of species and exact information on their salinity tolerance is essential for adequate ecological status assessment. In the present study, by using microscopy and metabarcoding, we investigated taxa of the genus Halamphora that are common in soda pans and soda lakes. We detected six species of which Halamphora dominici and H. veneta occurred frequently and often in high abundance (it was often dominant having relative abundance higher than 5%). Analyses of DNA data confirmed the separation of the two species; as a result, the reference database library has been supplemented with sequences of H. dominici. Furthermore, we have confirmed that this species, which is a significant indicator of sodic character, shows a positive correlation with salinity.

Animal-sourced model of human norovirus infection predicted using environmental DNA metabarcoding analysis

Human noroviruses (HNVs) are one of the major causes of food poisoning and severe gastroenteritis. However, prevention and medical treatment of HNV disease is underdeveloped due to the lack of ecological understanding of the HNVs including their host organisms. We address the potential source animals of HNVs based on the environmental DNA metabarcoding analysis. By focusing on the HNV-epidemic area in Japan, we found several bird species of ducks, pochards, carrion crow, and swan showing significant cross-correlation with the HNV detection from cultured oysters. Humans and livestock mammals do not exhibit correlation with HNVs. These results suggest an avian-sourced, non-human- or sewage-mediated model of HNV infection. Unraveling the unknown host animals of pathogens like HNV facilitates artificial propagation of the pathogen, leading to vaccine and small molecule drug development.

Enhancing the Conventional Culture: the Evaluation of Several Culture Media and Growth Conditions Improves the Isolation of Ruminal Bacteria

The rumen microbiota is critical in cattle digestion. Still, its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested comprised eight media cultures, three sample dilutions (10−2, 10−6, 10−12), and two incubation times (3 and 7 days). Bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates. The results indicate that none of the culture media recovered all rumen populations and that there was an altered relative abundance of the dominant phyla. In the rumen, Bacteroidetes and Firmicutes comprised 75% and 15% of the relative abundance, respectively, while in the culture media, these were 15% and 60%, respectively. Principal coordinate analysis (PCoA) of the bacterial community revealed significant shifts in population composition due to dilution, with 10−2 and 10−6 dilutions clustered closely while the 10−12 dilution differed markedly. In contrast, incubation duration did not influence population diversity. According to the results, two media, CAN and KNT, were selected based on their ability to recover more similar populations compared to the rumen sample. The metataxonomic study showed that CAN media had consistent reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate. From these, 64 pure cultures were obtained and 54 were identified through 16S rRNA gene sequencing. Being Streptococcus the most frequently isolated genus, this prevalence contrasts with the liquid media composition, underscoring the importance of refining single colony isolation strategies. Although no culture medium could replicate the native rumen bacterial population perfectly, our findings highlight the potential of CAN and KNT media in recovering populations that are more closely aligned to natural rumen conditions. In conclusion, our study emphasizes the importance of integrating molecular approaches in selecting suitable cultivation media and parameters to depict rumen bacteria accurately.

Exploring foraging preference of local fish species towards non-indigenous fouling communities near marinas: Insights from Remote Video Foraging System (RVFS) trials

Non-indigenous species (NIS) spread from marinas to natural environments is influenced by niche availability, habitat suitability, and local biotic resistance. This study explores the effect of indigenous fish feeding behaviour on NIS proliferation using fouling communities, pre-grown on settlement plates, as two distinct, representative models: one from NIS-rich marinas and the other from areas outside marinas with fewer NIS. These plates were mounted on a Remote Video Foraging System (RVFS) near three marinas on Madeira Island. After 24-h, NIS abundance was reduced by 3.5 %. Canthigaster capistrata's preference for marinas plates suggests potential biotic resistance. However, Sparisoma cretense showed equal biting frequencies for both plate types. The cryptogenic ascidian Trididemnum cereum was the preferred target for the fish. Our study introduces a global framework using RVFS for in-situ experiments, replicable across divers contexts (e.g., feeding behaviour, biotic resistance), which can be complemented by metabarcoding and isotopic analysis to confirm consumption patterns.

Correlation between microbial communities and volatile organic compounds in an urban soil provides clues on soil quality towards sustainability of city flowerbeds

Soil functionality is critical to the biosphere as it provides ecosystem services relevant for a healthy planet. The soil microbial composition is significantly impacted by anthropogenic activities, including urbanization. In this context, the study of soil microorganisms associated to urban green spaces has started to be crucial toward sustainable city development. Microbes living in the soil produce and degrade volatile organic compounds (VOCs). The VOC profiles may be used to distinguish between soils with various characteristics and management practices, reflecting variations in the activity of soil microbes that use a variety of metabolic pathways. Here, a combined approach based on DNA metabarcoding and GC-MS analysis was used to evaluate the soil quality from urban flowerbeds in Prato (Tuscany, Italy) in terms of microbial biodiversity and VOC emission profiles, with the final aim of evaluating the possible correlation between composition of microbial community and VOC patterns. Results showed that VOCs in the considered soil originated from anthropic and biological activity, and significant correlations between specific microbial taxa and VOCs were detected. Overall, the study demonstrated the feasibility of the use of microbe-VOC correlation as a proxy for soil quality assessment in urban soils.