A line with no hook: longline-associated passive eDNA samplers for deep-sea fish monitoring.

The Amazon basin harbors exceptional fish diversity, with more than 3,500 species reported. However, this biodiversity is increasingly threatened by anthropogenic activities and climate change. The Peruvian Amazon alone is home to nearly 1,000 freshwater fish species – approximately one-third of the entire Amazon – yet significant gaps remain in our understanding of their distribution, ecology, and conservation status. Molecular approaches, particularly environmental DNA (eDNA) metabarcoding, have emerged as promising alternatives for rapid and accurate biodiversity assessment. In this study, a metabarcoding workflow targeting a fragment of the 12S gene to eDNA samples collected from small streams and ponds near Iquitos, Peru, was applied to evaluate the applicability of this approach for local fish community inventories. Water from 12 sites was filtered, and DNA was extracted, amplified, and sequenced using high-throughput Illumina technology. Bioinformatic analyses included MOTU clustering, haplotype identification, and taxonomic assignment using the Lowest Common Ancestor (LCA) algorithm. A total of 226 amplicon sequence variants (ASVs) were identified, 95 of which were assigned to fish taxa and clustered into 44 MOTUs across four orders: Characiformes, Gymnotiformes, Siluriformes, and Cichliformes. The study highlights the effectiveness of eDNA metabarcoding in detecting both common and elusive species, while also highlighting current limitations due to incomplete DNA barcode reference libraries. These findings underscore the need to expand barcode databases to fully harness the potential of eDNA for monitoring Amazonian fish biodiversity and support its use as a valuable tool for inventorying fish communities in sensitive and understudied environments.

The Newport and Terrigal formations in sea cliffs north and south of Sydney yield well-preserved plant compressions and permineralized wood of late Early Triassic age (Spathian). The flora was a greenhouse spike vegetation at high palaeolatitude from early stages of plant adaptive radiation of the Mesozoic. The flora is best known for isoëtalean lycopsids, including Isoëtes, Tomiostrobus, Cylostrobus and Pleuromeia, which spread south with a global warming spike during the Spathian. Marattialean (Rienitsia), gleichenialean (Gleichenoides) and osmundalean (Todites) ferns may also reflect a spike of warmth and humidity. Schizoneura persisted from the Permian, but newly evolved Neocalamites was more like modern horsetails. Lepidopteris persisted from the Permian, but newly evolved seed ferns are represented by Dejerseya and Rochipteris. Newly evolved umkomasialean seed ferns dominate the flora, with large leaves of Dicroidium narrabeenense, D. gopadense, D. lancifolium, D. zuberi and Tetraptilon heteromerum, and wood of Kykloxylon. Large laminar structures of Umkomasia and Pteruchus are basal to the later Triassic adaptive radiation of smaller and more compact umkomasialean reproductive structures. The voltzialean conifer Voltziopsis angustum is more like Paleozoic than modern conifers. New taxonomic assignments include Gleichenoides narrabeenensis (Burges) comb. nov., Todites holmesii sp. nov., Rochipteris narrabeenensis (Walkom) comb. nov., and Kykloxlyon novae-valesiae (Burges) comb. nov. Gregory J. Retallack [gregr@uoregon.edu], Department of Earth Sciences, University of Oregon. Eugene, Oregon 97403, USA.

Species-level resolution is essential to understand gastric microbiome recovery after Helicobacter pylori eradication, yet short-read 16S rRNA approaches often obscure clinically relevant changes. Gastric biopsies from 121 adults in Bayan-Ölgii, Mongolia (71 H. pylori-positive, 50 H. pylori-negative) were analyzed, including nine paired pre- and post-eradication gastric biopsy samples collected six months apart, enabling exploratory longitudinal analysis. Full-length 16S rRNA (V1-V9) sequencing was performed using the Oxford Nanopore platform with EMU taxonomic assignment (SILVA v138.1/NCBI RefSeq). Ecological changes were evaluated using diversity indices, principal coordinates analysis (PCoA) with PERMANOVA, and differential abundance testing (DESeq2, FDR < 0.05). Eradication therapy (esomeprazole-bismuth-doxycycline-levofloxacin) achieved success in 54 of 57 H. pylori-positive patients (94.7%). H. pylori-positive microbiomes were dominated by H. pylori (91.8% ± 3.9%) and exhibited markedly reduced diversity (Shannon = 0.44 ± 0.11) compared with H. pylori-negative samples (2.08 ± 0.25; p < 0.001). Six months after eradication, diversity increased significantly (2.17 ± 0.20; p = 0.0001), with enrichment of oral commensals including Streptococcus mitis (↑ 11.9×), Neisseria elongata (↑ 13.7×), and Prevotella melaninogenica (↑ 13.0×). However, post-eradication profiles at six months remained distinct from H. pylori-negative communities (PERMANOVA R² = 0.12; p = 0.02). In total, 174 amplicon sequence variants changed significantly, including persistence of Fusobacterium nucleatum. Nanopore full-length 16S sequencing reveals fine-scale, clinically relevant shifts that are masked by partial-gene assays. Eradication rapidly restores microbial diversity, but at six months, is associated with a novel ecological equilibrium rather than complete normalization. This species-resolved approach offers a practical framework for post-eradication microbiome monitoring and may inform strategies to reduce residual gastric cancer risk in high-burden populations.

This study is the first attempt to inventory and evaluate the taxonomic identification of the Algerian myrmecofauna by integrating traditional morphology with molecular barcoding and phylogenetic approach. Sixty-one specimens of ants belonging to the family Formicidae, collected from different ecosystems in Algeria, were sequenced and barcoded using the mitochondrial cytochrome c oxidase subunit 1 gene (COX1). The DNA barcodes newly generated from this study were compared and analyzed with COX1 homologs available in GenBank databases representing putative conspecific and congeneric relatives of the Algerian ant samples and several others to represent the Formicoid, Poneroid, and Leptanillinae diversity to serve as molecular barcode references and outgroups for phylogenetic analyses. The results confirmed the COX1 gene as an excellent molecular marker at the generic level. They allowed the unambiguous taxonomic assignment of the Algerian specimens surveyed to fourteen Formicoid genera belonging to the Myrmicinae, Formicinae, and Dolichoderinae subfamilies, with abundance of Crematogaster, Tapinoma, Cataglyphis, and Componotus representatives. Such an integrative approach not only provided molecular support to most morphology-based identification but also allowed suggestion of taxonomic assignment or reassignment of some incompletely determined specimens, in accordance with the latest taxonomic revisions (e.g., Plagiolepis schmitzii, Camponotus spissinodis). It also helped to circumscribe some cases which need further investigations to illuminate complex evolutionary relationships and to re-evaluate and refine the taxonomic value of the most relevant diagnostic characters. Moreover, these preliminary data support the previous detection of three additional non-native ants in Algeria. Altogether, these results demonstrate the significant advantages that can be derived from a comprehensive development of such integrative studies for faster and better knowledge and understanding of the myrmecofauna diversity of Africa conserving regional biodiversity. Dataset published through GBIF (DOI: 10.15468/sjtjv2)

Environmental DNA (eDNA) metabarcoding is an innovative tool that is transforming ecological research. It offers a simple and effective method for simultaneously detecting numerous species across a wide range of environments. The method relies on assigning DNA sequences sampled from the environment to taxa, which is straightforward for species that have already been sequenced and are represented in reference databases. However, existing bioinformatics tools often fail to deliver accurate, fine-grained assignments when target species are absent from these databases. This limitation arises from handcrafted classification thresholds that do not account for nucleotide positional information. Here, we propose a deep neural architecture specifically designed to exploit both nucleotide identity and positional patterns in short TELEO sequences. Using an in-silico validation framework based on NCBI genbank sequences, we compare our approach with several state-of-the-art bioinformatics tools (Obitools, Kraken2, Lolo), as well as alternative sequence embedding methods, under controlled conditions. Our approach yields significantly higher classification accuracy at the genus and family levels, achieving average accuracies of 94.7% at the genus level and 86.5% at the family level, substantially outperforming the tested reference-based pipelines. The method remains robust with limited training data and shows improved performance when nucleotide positional information is preserved through sequence alignment. These results demonstrate the potential of AI-powered eDNA metabarcoding to complement existing taxonomic assignment tools, particularly in contexts where reference databases are incomplete or species-level resolution is not achievable, thereby supporting biodiversity monitoring and ecosystem management.

Successful applications of DNA barcoding rely on the accurate taxonomic identification of sequence fragments. When biological surveys with DNA barcoding target underexplored biological communities, sequence-based identification is often conducted using incomplete databases that do not fully cover the regional species pool. Consequently, specimens to be identified may include species not present in reference databases. Such unknown or "out-of-distribution" samples can cause misidentification if left undetected. A similarity cutoff is commonly used to detect out-of-distribution samples before taxonomic assignment, but its effectiveness has not been carefully studied. In this study, we evaluated the performance of out-of-distribution detection for DNA barcoding with genetic distance and deep learning metrics. Using extensively sampled datasets of multiple insect taxa, we measured the performance of identification and out-of-distribution detection under conditions in which genetic variations in species were sufficiently sampled. Although identification with DNA barcoding is a highly accurate process, even with short noisy fragments, out-of-distribution detection was more susceptible to a reduction in performance due to sequence noise and a lack of diagnosable characters. When fragments shorter than 300 bp were used for out-of-distribution detection, large performance reductions were observed irrespective of detection methods. Our results provide guidelines for designing unknown-proof identification procedures by determining factors affecting out-of-distribution detection performance.

Tannery effluents constitute highly complex chemical and biological matrices that can affect ecosystem integrity and public health. In Paraguay, metagenomic information on industrial discharge remains limited. In this context, the aim of this study was to characterize microbiome diversity and detect antibiotic resistance genes (ARGs) via metagenomic sequencing complemented by chemical analyses. Total DNA was sequenced using Oxford Nanopore technologies and analyzed with Kraken2 for taxonomic assignment and CARD for ARG detection. The results revealed a hypersaline, metal-containing effluent with a high organic load and measurable nitrogen and phosphorus concentrations. Microbiome profiles were dominated by Pseudomonadota (77.2%), primarily Thiocapsa (27.8%) and Francisella (23.0%). The phototrophic and sulfur-oxidizing metabolism characteristic of Thiocapsa may explain the distinctive coloration of the effluent, while the predominance of Francisella is consistent with tolerance to hostile environmental conditions. DNA sequences assigned to taxa of clinical relevance, including Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella pneumoniae, were also detected, along with a range of ARGs associated with resistance to tetracyclines, β-lactams, and aminoglycosides. These findings demonstrate that treated tannery effluent can retain clinically relevant genetic material and ARGs, underscoring the need to integrate metagenomic surveillance into environmental monitoring frameworks to better understand and mitigate emerging resistance determinants in aquatic systems. This study provides one of the first metagenomic characterizations of a tannery effluent in the country and contributes novel insights at a regional scale.

BackgroundCtenocephalides felis is a common ectoparasite of dogs and cats and can transmit a variety of pathogens including Bartonella and Rickettsia species. These bacteria, along with the known endosymbiont Wolbachia, are well-documented members of the C. felis microbiome, but species-level information is limited. Additionally, little is known about the variation in the C. felis microbiome in fleas from different sources and when different sequencing methods are applied to the same samples.ObjectiveThis study aimed to characterize the flea microbiome using both short-read (V3/V4) and long-read (full-length) 16S rRNA gene sequencing, determine whether long-read sequencing improves species-level identification especially in known pathogenic genera, and evaluate differences in microbial composition between fleas collected from cats, dogs, and environmental traps.MethodsFleas were collected from cats, dogs, and traps in flea-infested homes in Florida, pooled by source, and sequenced using short- (V3/V4) and long-read (full-length) 16S rRNA gene sequencing. Microbial prevalence and abundance were compared across sequencing approaches. Community composition was evaluated for differences between sources and houses. Candidate members of the flea microbiome were identified based on a combination of prevalence, abundance, and statistical signatures of potential contaminant origin. For Rickettsia and Bartonella, species-level taxonomic assignments were refined using a phylogenetic approach.ResultsWolbachia, Rickettsia, and Bartonella were the most prevalent and abundant taxa. Spiroplasma was identified as a fourth core member of the flea microbiome. Long-read sequencing enabled better, but not perfect, species-level classification of Bartonella and Rickettsia compared to short-read sequencing. Important relationships between specific ASVs and flea sources were identified, for example fleas from cats harbored higher abundances of B. clarridgeiae and B. henselae than fleas from traps.

We assessed the fungal diversity and functional profile of two soils collected in contrasting environments: one unimpacted soil, Hennequin Point, King George Island, and the other impacted by whale oil, Whalers Bay, Deception Island, Maritime Antarctica, using metagenomic approaches. Taxonomic assignment revealed a predominance of Ascomycota in both soils. A total of 20 and 23 fungal genera were identified at King George and Deception islands, respectively. The rare genera Thermothielavioides, Pyricularia, Fulvia, and Coccidioides were detected in the Antarctic environment. The highest fungal diversity was observed in the soil of Deception Island. Canonical analysis of King George Island soil displayed higher values of total organic carbon, sulfur, and lead, which may have favored the presence of the genera Puccinia, Lachancea, and Akanthomyces. The soil of Deception Island presented correlations with higher levels of nitrogen, chromium, and iron, with a predominance of genera such as Aspergillus, Trichoderma, and Malassezia. Functional analysis revealed distinct adaptive strategies among the soils. Domains related to translation, gene regulation, and metabolic efficiency were observed for fungi in Hennequin Point soil, King George Island, suggesting resource optimization in a cold, moss-covered environment. In Deception Island soil, fungal redox metabolism, iron acquisition, and the degradation of nitrogen compounds were highlighted, reflecting adaptation to an anthropogenic soil rich in metal oxides. Both soils exhibited functional fungal networks involved in hydrolytic enzymatic pathways that may act in the decomposition of organic compounds. New sequencing must be performed due to the insufficient depth of the data. Our results indicated that the soil from Hennequin Point and Whalers Bay exhibited distinct fungal communities, which can be influenced by environmental and ecological factors such as moss, oil, and heavy metals encountered in pristine and oil-impacted soils resulting from anthropogenic activities over the years.

Abstract BACKGROUND Ulcerative colitis (UC) is a chronic inflammatory bowel disease driven by complex interactions between genetics, environment, and the gut microbiota, with rising incidence in developing nations. India, currently in stage 2 of the IBD epidemiological transition, reports one of the highest case burdens in Southeast Asia. While gut microbial dysbiosis is implicated in UC pathogenesis, most studies have focused on fecal microbiota, with limited exploration of the mucosa-associated microbiome (MAM), which resides in close contact with the intestinal epithelium. This study aimed to characterize microbial signatures relevant to UC pathogenesis in Indian patients, where high fecal–oral transmission and infection rates may uniquely shape microbial dynamics. METHODS The microbiomes of colonic mucosal biopsies were characterized from 30 patients with established UC (inflamed areas) and 30 non-IBD controls (normal mucosa). DNA was extracted using a mechanical–chemical lysis protocol followed by kit-based purification. V3–V4 region of 16S rRNA gene was sequenced on the Illumina MiSeq platform (2 × 300 bp). Data were processed using the DADA2 pipeline, followed by taxonomic assignment with the SILVA database. Diversity analyses (alpha and beta), phylum-level comparisons, LEfSe-based biomarker discovery, species-level differential abundance testing, and Spearman correlation-based co-occurrence network analysis were conducted to assess the microbial communities in UC. RESULTS UC samples showed significantly altered beta diversity across all taxonomic levels and a notably elevated Firmicutes-to-Bacteroidota ratio (mean 1.91). Enrichment of Firmicutes and Desulfobacterota, alongside reduction of Fusobacteriota, Actinobacteriota, Patescibacteria, and Spirochaetota, characterized the UC microbiome. LEfSe revealed UC-associated families such as Campylobacteraceae, Lachnospiraceae, and Cardiobacteriaceae, while Porphyromonadaceae, Spirochaetaceae, and Actinomycetaceae were enriched in controls. At the species level, pathobionts including Bacteroides fragilis, Parabacteroides goldsteinii, Escherichia sp., and Campylobacter concisus were elevated in UC, whereas health-associated commensals like Streptococcus infantis, Capnocytophaga spp., and Lactobacillus casei were reduced. Network analysis revealed strong positive correlations among pro-inflammatory taxa in UC, indicating tightly clustered microbial communities potentially driving mucosal inflammation. CONCLUSIONS Our findings highlight distinct mucosal bacterial signatures in Indian patients with UC, marked by enrichment of pro-inflammatory pathobionts, depletion of beneficial commensals, and an increased Firmicutes-to-Bacteroidota ratio. These results underscore the importance of region-specific MAM profiling and support the integration of microbial signatures into personalized treatment strategies for UC.

Introduction. The foliar structure of banana plants is a determining factor in the yield and development of the crop. The microbial composition in foliar spots is relevant for understanding the emergence and spread of diseases. Objective. To perform the isolation and molecular identification of fungi in banana plants with and without leaf spots. Materials and Methods. In 2019, a non-experimental research study was conducted with a quantitative approach and descriptive level, using banana leaves from Musa acuminate (cv.IC2) with spots (HCM) and without spots (HSM), collected from a conventionally managed agricultural plot in northern Peru. Leaf fragments of 5 x 5 mm were cultivated on microbiological media, and fungal strains were isolated based on their macrostructures. DNA was extracted from the strains, and conventional PCR was performed targeting the ITS region of fungi (700 bp). The PCR products were then sequenced using the Sanger method in double strands. Taxonomic assignments at the species level were made using the BLAST tool, based on homology comparison with sequences from GenBank. Results. A total of eleven fungal species were identified in HCM and eight in HSM, including both phytopathogenic and non-phytopathogenic species. The predominant species in HCM were Fusarium spp., Cladosporium cladosporioides, and Lasiodiplodia theobromae. Additionally, it was observed that HCM shared three genera with HSM: Nigrospora, Cladosporium, and Fusarium. Conclusion. Fungal isolation and molecular identification were performed on bananas with and without leaf spots, and phytopathogenic and beneficial species were found.

This study aimed to compare the composition and diversity of the salivary microbiome in stunted and nonstunted children using 16S rRNA gene sequencing to explore the relationship between nutritional status and oral microbiota.A total of 20 saliva samples were collected from children aged 6 to 10 years, comprising two groups: stunted (n = 10) and healthy controls (n = 10). Deoxyribonucleic acid was extracted, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced. Bioinformatics analysis included taxonomic assignment, calculation of relative abundance, α diversity (using Shannon and Simpson indices), β diversity (UniFrac-based principal coordinate analysis and permutational multivariate analysis of variance [PERMANOVA]), and differential abundance testing using the Mann-Whitney U test.The dominant phyla in both groups were Proteobacteria, Firmicutes, and Bacteroidota, with Proteobacteria being more prevalent in the stunted group. At the genus level, Neisseria and Veillonella were more abundant in stunted children. Notably, Veillonella was significantly elevated in the stunted group (28.6%) compared with controls (14.9%, p = 0.0376). Alpha diversity indices revealed a higher diversity trend in the stunted group, although this difference was not statistically significant (Shannon, p = 0.130; Simpson, p = 0.762). Beta diversity analysis revealed no considerable clustering between groups (PERMANOVA p > 0.05), indicating moderate interindividual variability but no clear group separation.Children with stunted growth demonstrated distinct microbial signatures in their salivary microbiota, particularly in the increased abundance of Proteobacteria and Veillonella, suggesting a potential link between chronic undernutrition and oral microbial dysbiosis. These findings underscore the need for additional studies to investigate the impact of nutritional status on oral and systemic health through the microbiome axis.

Abstract Lenoks, species within the genus Brachymystax, are freshwater salmonids with a scattered distribution in the rivers of Siberia, Northeast China, Xinjiang, Hebei, and the Qinling Mountains. Owing to long-term population declines, all species assigned to Brachymystax are protected by law in China. However, the evolutionary history and species-level systematics of this genus remain controversial, complicating taxonomic designations and conservation efforts. In particular, the geographical separation of populations may have resulted in the formation of phenotypically similar cryptic species. We built a chromosome-level genome assembly of B. tsinlingensis and re-sequenced the genomes of 103 individuals of Chinese Brachymystax spp. from five geographically isolated locations. Population genomic and phylogenomic analyses based on nuclear single nucleotide polymorphisms (SNPs) and mitochondrial genomes revealed six different genetic lineages, of which at least one, the Hebei lineage, represents a cryptic species. Notably, the results suggest that the sympatric species B. lenok and B. tumensis are not close relatives, but the former is more closely related to the new species B. sp. Xinjiang with an estimated divergence time of c. 630 Ka, indicating that closely related sympatric species may not have evolved via sympatric speciation in areas influenced by Pleistocene climate changes. We observed mito-nuclear phylogenomic discordance in Brachymystax caused by the strong gene flow between B. lenok and B. tumensis. Phylogenetic and demographic analyses emphasize the important role of interglacial refugia in promoting speciation and underscore the impact of historical gene flow. Compared with other lenoks, the Gansu population had the lowest genetic diversity, suggesting that particular attention to protect its genetic resources may be required. Finally, we suggest that cross-regional proliferation and release of lenoks should be banned in the future to protect the genetic integrity of these divergent groups.

Viruses, as a population of subcellular entities of genetic material in a package, are at least an order of magnitude as numerous as that of their cellular hosts, on which they depend. While the particulars of the emergence of the ancestors of viruses are unknown, the evolutionary histories of their genes are possible by the evolutionary methods of gene sequence comparison and phylogenetic reconstruction. These studies depend on nucleotide or protein sequence data and a reference to the underlying process of genetic replication, which is a cause of taxon-specific differences in the rates of genetic mutation and correction of replication errors. Viral types also do not easily fit within the confines of traditional typologies, such as the use of a host preference for a biological marker of their taxonomic assignments. Instead, their population processes are dynamic and vary across taxa, which are causes of genetic change and new viral lineages. Since a naive deterministic model of evolution is not supported in the corpus of modern science, it is instead an ideal to infer knowledge of viral origins by models based on the stochastic processes of molecular evolution. These approaches are the basis for validating and building knowledge on virus evolution that leads to confidence and robustness in scientific findings (a requisite to the formation of a scientific belief).

Ecosystems associated with retreating glaciers are undergoing rapid transformation in the context of a changing climate. Invertebrate fauna, including the Chironomidae insect family (non-biting midges), is part of the active biology that characterizes glaciers and their surrounding habitats. The underlying microbiology, present in both the insects’ guts and their physical habitat, represents a critical interface, controlling, on the one hand, the basis of nutrient geochemical cycling and, on the other, the health and nutritional physiology of its hosts. We aimed to assess the extent to which insect-borne bacteria resemble those found in icemelt water and the surrounding wet and terrestrial environments, in order to determine also whether the bacteria found associated with the insects could be interpreted mainly as specific dwellers, putatively involved with active physiological functions, or also as transient cells taken in for other purposes. To this end, we analyzed physical and biological samples from ten different chironomid species in two glacier-fed streams, one proglacial pond, and the surrounding habitats of three glacier systems (Agola, Amola and Mandrone) in the Italian Alps. The samples were analyzed using culture-independent amplified 16S rRNA gene bacterial metabarcoding sequencing. The bacterial diversity in glacial habitats was unexpectedly high, with numbers of sequence variants similar to those recorded in temperate, lowland, productive soils, and almost seven times higher than those found in insects. There was minimal coincidence in sequence variants between insects and habitats, amounting to just 4.9% shared cases, and the few taxa found in both insects and habitats were mostly overrepresented in the former. Additionally, there were no significant differences between insect species or between insect communities from different sites. A number of taxa occurring uniquely in insects or habitats showed peculiarities at all taxonomic levels, including specific phyla. Evidence of microevolutionary distinctness was observed in the form of sequence variants assigned to the same taxonomic name that differed in specific sequence bases and were found to be partitioned either in insect or environmental samples. Interestingly, sequence variants found in both insects and environments scored higher in bioinformatic identification, reaching deeper assigned ranks compared to variants occurring only in insects or the environment. In essence, the compared insect and environmental communities showed a very low level of symmetry and consequently a very high level of specificity to one or the other condition. The data provided limited support about the diet of chironomids in relation to the microbiota of the habitat. This suggests that the food resource for these insects likely consists mostly of dissolved organic matter and detritus of various origins, rather than intact bacteria with sequenceable genomes, as is the case elsewhere with ruminant herbivores or birds.

Accurate profiling of soil and root-associated bacterial communities is essential for understanding ecosystem functions and improving sustainable agricultural practices. Here, a comprehensive, modular workflow is presented for the analysis of full-length 16S rRNA gene amplicons generated with Oxford Nanopore long-read sequencing. The protocol integrates four standardized steps: (i) quality assessment and filtering of raw reads with NanoPlot and NanoFilt, (ii) removal of plant organelle contamination using a curated Viridiplantae Kraken2 database, (iii) species-level taxonomic assignment with Emu, and (iv) downstream ecological analyses, including rarefaction, diversity metrics, and functional inference. Leveraging high-performance computing resources, the workflow enables parallel processing of large datasets, rigorous contamination control, and reproducible execution across environments. The pipeline’s efficiency is demonstrated on full-length 16S rRNA gene datasets from yellow pea rhizosphere and root samples, with high post-filter read retention and high-resolution community profiles. Automated SLURM scripts and detailed documentation are provided in a public GitHub repository (https://github.com/henrimdias/emu-microbiome-HPC; release v1.0.2, emu-pipeline-revised) and archived on Zenodo (DOI: 10.5281/zenodo.17764933).Key features• Implement rigorous quality control (QC) of raw 16S rRNA Nanopore reads and sequencing controls.• Remove plant organelle contamination with a curated Kraken2 database.• Perform high-resolution taxonomic assignment of full-length 16S rRNA reads using Emu.• Integrate downstream statistical analyses, including rarefaction, PERMANOVA, and DESeq2 differential abundance.• Conduct scalable microbiome diversity and functional analyses with FAPROTAX.

The Mediterranean Sea is one of the major reservoirs of marine biodiversity and harbors an exceptionally high number of endemic species. Nevertheless, the taxonomic status of many of these endemics still requires confirmation through integrative, modern approaches. We hereby first investigated the identity and status of a rare and endemic box crab, known either as Calappa rosea or Calappa rissoana , through a multidisciplinary approach that capitalized on the use of nomenclatural rules, museology, integrative taxonomic approaches (morphology, complete mitochondrial genomes, nuclear markers, and phylogenetic analyses), and passive citizen science. Almost all morphological characters failed to differentiate it from the related species Calappa granulata , whereas differences in coloration were mostly confirmed here. However, all molecular approaches supported the conspecificity of these two taxa. The combined use of passive citizen science and statistical analyses revealed that the formerly endemic species is an unrecognized ontogenetic stage of C. granulata , and, in particular, a rare transitional phase that connects early juveniles to fully developed adults. This renders the investigated taxon a new junior synonym of C. granulata and solves nomenclatural and taxonomic ambiguities related to native Mediterranean box crabs, which have remained unsettled for over 200 years. The present study, therefore, provides the first comprehensive reconstruction of the ontogenetic changes occurring in the common Mediterranean box crab throughout its various life stages and raises questions on whether these transitions occur worldwide across box crabs or are restricted to a few species. Finally, it presents new, conspicuous, and detailed morphological and molecular data on the type species of the genus Calappa , facilitating future phylogenetic reconstructions and taxonomic assignments within the entire family Calappidae, and discusses the putative occurrence of the other box crabs in the area, suggesting a critical re‐evaluation of all historical data and records.

Ecosystems associated with retreating glaciers are undergoing rapid transformation in the context of a changing climate. Invertebrate fauna, including the Chironomidae insect family (non-biting midges), is part of the active biology that characterizes glaciers and their surrounding habitats. The underlying microbiology, present in both the insects' guts and their physical habitat, represents a critical interface, controlling, on the one hand, the basis of nutrient geochemical cycling and, on the other, the health and nutritional physiology of its hosts. We aimed to assess the extent to which insect-borne bacteria resemble those found in icemelt water and the surrounding wet and terrestrial environments, in order to determine also whether the bacteria found associated with the insects could be interpreted mainly as specific dwellers, putatively involved with active physiological functions, or also as transient cells taken in for other purposes. To this end, we analyzed physical and biological samples from ten different chironomid species in two glacier-fed streams, one proglacial pond, and the surrounding habitats of three glacier systems (Agola, Amola and Mandrone) in the Italian Alps. The samples were analyzed using culture-independent amplified 16S rRNA gene bacterial metabarcoding sequencing. The bacterial diversity in glacial habitats was unexpectedly high, with numbers of sequence variants similar to those recorded in temperate, lowland, productive soils, and almost seven times higher than those found in insects. There was minimal coincidence in sequence variants between insects and habitats, amounting to just 4.9% shared cases, and the few taxa found in both insects and habitats were mostly overrepresented in the former. Additionally, there were no significant differences between insect species or between insect communities from different sites. A number of taxa occurring uniquely in insects or habitats showed peculiarities at all taxonomic levels, including specific phyla. Evidence of microevolutionary distinctness was observed in the form of sequence variants assigned to the same taxonomic name that differed in specific sequence bases and were found to be partitioned either in insect or environmental samples. Interestingly, sequence variants found in both insects and environments scored higher in bioinformatic identification, reaching deeper assigned ranks compared to variants occurring only in insects or the environment. In essence, the compared insect and environmental communities showed a very low level of symmetry and consequently a very high level of specificity to one or the other condition. The data provided limited support about the diet of chironomids in relation to the microbiota of the habitat. This suggests that the food resource for these insects likely consists mostly of dissolved organic matter and detritus of various origins, rather than intact bacteria with sequenceable genomes, as is the case elsewhere with ruminant herbivores or birds.

Abstract Four new species of Poecilosclerida (Porifera, Demospongiae) assigned to the genera Latrunculia and Iophon are described from South Africa and Namibia, located in the Namaqua ecoregion. The Porifera occurring along the continental shelf within this ecoregion are relatively well-known, with 76 species formally described in previous literature. Of these, 35 species belong to the Order Poecilosclerida. Additionally, Latrunculia ( Aciculatrunculia ) biformis is reported from the continental shelf on the west coast of South Africa, extending its range further northwards into the South Atlantic. DNA barcoding and molecular phylogenetic analyses were employed to ensure accurate taxonomic assignment and designation of new species.