Barcode Sequences Research Articles

Environmental niche models improve species identification in DNA barcoding

Abstract Recent advances in DNA barcoding have immeasurably advanced global biodiversity research in the last two decades. However, inherent limitations in barcode sequences, such as hybridization, introgression or incomplete lineage sorting can lead to misidentifications when relying solely on barcode sequences. Here, we propose a new Niche‐model‐Based Species Identification (NBSI) method based on the idea that species distribution information is a potential complement to DNA barcoding species identifications. NBSI performs species membership inference by incorporating niche modelling predictions and traditional DNA barcoding identifications. Systematic tests across diverse scenarios show significant improvements in species identification success rates under the newly proposed NBSI framework, where the largest increase is from 4.7% (95% CI: 3.51%–6.25%) to 94.8% (95% CI: 93.19%–96.06%). Additionally, obvious improvements were observed when using NBSI on potentially ambiguous sequences whose genetic nearest neighbours belongs to another species or more than two species, which occurs commonly with species represented by single or short DNA barcodes. These results support our assertion that environmental factors/variables are valuable complements to DNA sequence data for species identification by avoiding potential misidentifications inferred from genetic information alone. The NBSI framework is currently implemented as a new R package, ‘NicheBarcoding’, that is open source under GNU General Public Licence and freely available from https://CRAN.R‐project.org/package=NicheBarcoding.

Sympatric diversity pattern driven by the secondary contact of two deeply divergent lineages of the soybean pod borer Leguminivora glycinivorella.

The soybean pod borer, Leguminivora glycinivorella (Matsumura), is an important tortricid pest species widely distributed in most parts of China and its adjacent regions. Here, we analyzed the genetic diversity and population differentiation of L. glycinivorella using diverse genetic information including the standard cox1 barcode sequences, mitochondrial genomes (mitogenomes), and single-nucleotide polymorphisms (SNPs) from genotyping-by-sequencing. Based on a comprehensive sampling (including adults or larvae of L. glycinivorella newly collected at 22 of the total 30 localities examined) that covers most of the known distribution range of this pest, analyses of 543 cox1 barcode sequences and 60 mitogenomes revealed that the traditionally recognized and widely distributed L. glycinivorella contains two sympatric and widely distributed genetic lineages (A and B) that were estimated to have diverged ∼1.14 million years ago during the middle Pleistocene. Moreover, low but statistically significant correlations were recognized between genetic differentiation and geographic or environmental distances, indicating the existence of local adaptation to some extent. Based on SNPs, phylogenetic inference, principal component analysis, fixation index, and admixture analysis all confirm the two divergent sympatric lineages. Compared with the stable demographic history of Lineage B, the expansion of Lineage A had possibly made the secondary contact of the two lineages probable, and this process may be driven by the climate fluctuation during the late Pleistocene as revealed by ecological niche modeling.

Molecular identification and antimicrobial resistance patterns of enterobacterales in community urinary tract infections among indigenous women in Ecuador: addressing microbiological misidentification

BackgroundAntibiotic resistance of Enterobacterales poses a major challenge in the treatment of urinary tract infections (UTIs). In low- and middle-income countries (LMICs), standard microbiological (i.e. urine culture and simple disk diffusion test) methods are considered the “gold standard” for bacterial identification and drug susceptibility testing, while PCR and DNA sequencing are less commonly used. In this study, we aimed to re-identifying Enterobacterales as the primary bacterial agents responsible for urinary tract infections (UTIs) by comparing the sensitivity and specificity of traditional microbiological methods with advanced molecular techniques for the detection of uropathogens in indigenous women from Otavalo, Ecuador.MethodsA facility-based cross-sectional study was conducted from October 2021 to February 2022 among Kichwa-Otavalo women. Pathogens from urine samples were identified using culture and biochemical typing. Morphological identification was doble-checked through PCR and DNA sequencing of 16S, recA, and rpoB molecular barcodes. The isolates were subjected to antimicrobial susceptibility-testing using disk diffusion test.ResultsThis study highlighted a 32% misidentification rate between biochemical and molecular identification. Using traditional methods, E. coli was 26.19% underrepresented meanwhile Klebsiella oxytoca was overrepresented by 92.86%. Furthermore, the genera Pseudomonas, Proteus, and Serratia were confirmed to be E. coli and Klebsiella spp. by molecular method, and one Klebsiella spp. was reidentified as Enterobacter spp. The susceptibility profile showed that 59% of the isolates were multidrug resistant strains and 31% produced extended spectrum beta-lactamases (ESBLs). Co-trimoxazole was the least effective antibiotic with 61% of the isolates resistant. Compared to previous reports, resistance to nitrofurantoin and fosfomycin showed an increase in resistance by 25% and 15%, respectively.ConclusionsCommunity-acquired UTIs in indigenous women in Otavalo were primarily caused by E. coli and Klebsiella spp. Molecular identification (16S/rpoB/recA) revealed a high rate of misidentification by standard biochemical and microbiological techniques, which could lead to incorrect antibiotic prescriptions. UTI isolates in this population displayed higher levels of resistance to commonly used antibiotics compared with non-indigenous groups. Accurate identification of pathogens causing UTIs and their antibiotic susceptibility in local populations is important for local antibiotic prescribing guidelines.

First detection of the obligate biotroph Phytophthora cyperi causing disease on yellow nutsedge in the United States, and establishment of molecular barcoding sequences for the species

A pathogen causing disease on yellow nutsedge (Cyperus esculentus) growing in and near irrigated areas of onion seed and bulb fields in Idaho and Oregon during July and August, 1996 was morphologically and molecularly characterized. Plant symptoms included necrotic spots, blotches and blighting of leaves, stems (culms) and bracts. Symptomatic tissue contained glistening, white masses of mycelium and oval, semi-papillate sporangia emerging from stomata on sporangiophores. Later, subspherical oogonia with smooth oospores were found embedded in older, infected tissue. Phytophthora cyperi was identified as the causal agent and pathogenicity was confirmed by inoculating healthy nutsedge plants with sporangia from infected plants. Infected samples tested negative using Agdia’s Phytophthora ELISA kit and all efforts to culture the organism on semi-selective or general fungal media failed. To aid future efforts to detect this species, sequences of the two primary barcoding loci, ITS rDNA and mitochondrial cox1 were obtained from the organism and analyzed, finding an unexpectedly large ITS sequence characterized by a ~1300 bp insertion.

Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq.

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.

P4.07E.08 Single-Cell TCR Barcoding and RNA Sequencing Illuminate Distinct Tumor-Draining Lymph Node T Cell Responses to Neoadjuvant PD-1 Blockade

P4.07E.08 Single-Cell TCR Barcoding and RNA Sequencing Illuminate Distinct Tumor-Draining Lymph Node T Cell Responses to Neoadjuvant PD-1 Blockade

Studies on Neotropical Pseudophyllinae: Integrative description of Dentotibium gen. nov., and of a toothed foretibia katydid (Orthoptera: Tettigoniidae) that sings from burrows in branches

Herein, we describe the genus Dentotibiumgen. nov. and the species D. ramucantorissp. nov, a toothed foretibia katydid within the subfamily Pseudophyllinae from Colombia. This new katydid is a forest dweller inhabiting oak woodlands between 1600 and 2300 m above sea level, only found in the western slopes of the Eastern Andes of Boyacá Department, Colombia. This new species burrows in woody branches and shows big heads in both sexes. The appearance of Dentotibiumgen. nov. is similar to that of Trichotettix Stål, 1873 but differs in having tubercles over the dorsal margin of the foretibiae. It is also close to Nastonotus Bolívar, 1890, but differs from in lacking the three black stripes in the vertex and having a less marked frontal suture and smoother face. Males sing all night long, and their calling song consists of periodic syllables with three differentiable phases. Seasonal calling activity lowers remarkably during the dry season (Jun–Aug). DNA barcoding sequences for the paratypes made available in a previous study are included here.

DNA Barcoding and New Records of Water Mites (Acari, Hydrachnidia) from Bosnia and Herzegovina

During the last decade, DNA barcoding has become a widely applicated approach in the identification of water mite species. Still, our knowledge about the molecular diversity and number of DNA barcodes of water mite species that inhabit Bosnia and Herzegovina is limited. In this study a total of 50 mitochondrial barcode sequences were obtained from 18 water mite species collected in Bosnia and Herzegovina. Seven species, i.e. Sperchon violaceus Walter, 1944, S. milisai Pešić, 2022, Lebertia glabra Thor, 1897, L. reticulata (Koenike, 1919), Torrenticola lukai Pešić, Valdecasas & Garcia-Jimenez, 2012, Hygrobates turcicus Pešić, Esen & Dabert, 2017, and Arrenurus cylindratus Piersig, 1896 are added as new for the water mite fauna of Bosnia and Herzegovina. Our results revealed that all recorded water mite species in our study could be identified when we consider global and regional DNA barcode database.

Anomalous latitudinal gradients in parasitoid wasp diversity-Hotspots in regions with larger temperature range.

Knowledge of global patterns of genetic diversity is essential for biodiversity conservation as this parameter describes the ability of a species to respond to environmental changes. Ichneumonoids parasitoid wasps are among the few taxa showing an anomalous latitudinal diversity gradient. Using the largest georeferenced molecular dataset for this group, we used a macrogenetics approach to examine latitudinal patterns and predictors of intraspecific genetic diversity. We calculated the mean nucleotide diversity of mitochondrial DNA barcode sequences at three geographic levels: grid cells, latitudinal bands and climatic zones. Nucleotide diversity values were consistently higher at northern temperate latitudes, peaking at 50°. We found a positive but weak relationship between intraspecific diversity and the latitude, between intra- and interspecific diversity, and a positive effect of the temperature range. Examining the spatial relationship between different levels of biodiversity and its drivers is particularly relevant considering climate change and its impact on species distribution. Yet, in insects, it has been challenging to integrate ecological, evolutionary and geographical components when analysing the processes leading to species richness gradients.

Mapping multiple RNA modifications simultaneously by proximity barcode sequencing.

RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m 6 A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m 6 A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m 6 A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m 6 A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m 6 A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies.

Integrative taxonomic review of the genus Rhodostrophia Hübner, 1823 and its allied genus Tanaotrichia Warren, 1893 (Lepidoptera: Geometridae) from the Western Himalaya

The genus Rhodostrophia Hübner, 1823 and its close ally Tanaotrichia Warren, 1893 (Geometridae: Sterrhinae) are reviewed from the North Western and Trans Himalayan regions of the Indian Himalaya along with some specimens from the Pakistan. In total, eight species were studied—seven Rhodostrophia spp. and one species of Tanaotrichia—with detailed morphological and genital characteristics. Rhodostrophia borealis stat. n. is raised from subspecies of R. cinerascens to species level after studying external morphology and distinct genitalia-based diagnostic features. However, this warrants further validation following the barcode sequencing of R. cinerascens. Illustrations of habitus, including type specimens, are provided along with images of genitalia and the sequence data of the mitochondrial cytochrome c oxidase subunit I (COI 5′ terminus) gene. A checklist of Indian Rhodostrophia and Tanaotrichia species is included with information on their documented distributional range.

Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.

Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

High-throughput protein characterization by complementation using DNA barcoded fragment libraries.

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq's utility is demonstrated, with future applications in ecology, health, and engineering.

Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife

The surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics.

Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions.

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of the erm-bar DNA Basic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeri Alternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strains Basic Protocol 3: Setting up mixed populations of barcode-tagged strains Basic Protocol 4: Performing a competitive growth assay Basic Protocol 5: Amplicon library preparation and equimolar pooling Basic Protocol 6: Sequencing on Illumina iSeq 100 Basic Protocol 7: BarSeq data analysis.

Morphological and molecular characterisation of Mordellistenapeloponnesensis Batten, 1980 (Coleoptera, Mordellidae), with first records from Italy and Turkey.

Mordellistenapeloponnesensis Batten, 1980, previously known from Cyprus and Greece, is reported from Italy and Turkey for the first time. The species is redescribed based on type specimens and additional material from its entire known distributional range. Eighteen DNA barcoding sequences of M.peloponnesensis from Greece, Cyprus, and Italy were generated, and genetic variability across the sampling localities was examined. Three mitochondrial haplotypes were detected within M.peloponnesensis. Specimens from mainland Italy share the same haplotype as those from Rhodes and Cyprus, whereas Sardinian specimens exhibit a distinct haplotype. The third haplotype is represented by one specimen from Cyprus. The DNA barcoding sequences of M.peloponnesensis were compared with those of the morphologically allied M.gemellata Schilsky, 1898, and M.pyrenaea Ermisch, 1966, to reveal the phylogenetic relationships between the species.

Updated checklist with new records and molecular data for the mosquitoes (Diptera: Culicidae) of Hong Kong.

An extensive mosquito survey was carried out in Hong Kong from September to October 2022, employing a variety of collection methods. Specimens were identified using a combination of morphology and mitochondrial cytochrome C oxidase subunit 1 (COI) barcode sequences. Twenty-nine species, including three new records, i.e., Culex bicornutus (Theobald), Culex cinctellus Edwards, and Lutzia chiangmaiensis Somboon & Harbach, were collected. Phylogenetic analysis of COI sequences of Culex annulus Theobald and Culex vishnui Theobald collected in Hong Kong and elsewhere revealed that the sequences of the two nominal species are genetically very similar and are included in the same clade. Consequently, the synonymy of Cx. annulus with Cx. vishnui is reinstated. Lutzia halifaxii (Theobald) is removed from the list of species in Hong Kong and is replaced with Lutzia vorax Edwards, the identification of which is confirmed in the present study. The record of Culex spiculothorax Bram recorded in Hong Kong is replaced with the senior synonym Culex sasai Kano, Nitahara & Awaya. The occurrence of Anopheles fluviatilis James and Aedes aegypti (Linnaeus) is discussed. Finally, an updated checklist of the mosquitoes of Hong Kong, which now includes 76 species representing 14 genera, is provided, with notation of those species that vector pathogens of human diseases.

Bulk RNA Barcode Sequencing Reveals Role of RNA Splicing in Aging Dermal Stem Cell Modulation by a Botanical Extract

Skin aging is a complex, multifaceted process influenced by both intrinsic and extrinsic factors. Understanding the molecular mechanisms underlying skin aging is crucial for developing effective anti-aging strategies. Dermal stem cells play a pivotal role in maintaining skin homeostasis, but their functionality is compromised with aging. This study investigated the impact of aging on dermal stem cells and explored the potential of natural extracts in modulating their biological characteristics. Using bulk RNA barcoding and sequencing (BRB-seq), we identified differentially expressed genes (DEGs) between young and aged dermal stem cells, revealing alterations in cellular processes, including cell proliferation, ECM synthesis, and RNA splicing. We also demonstrated that a natural extract, comprising callus cells and Alpine rose leaf extracts, influenced RNA splicing in aged dermal stem cells, leading to improved dermal structure and integrity in vitro. Our findings suggest that natural extracts may exert their effects through senolytic activity and the modulation of RNA splicing, a process crucial to gene expression and cellular function. This study underscores the potential of integrating high-throughput transcriptomics in understanding skin aging, presenting new avenues for the development of innovative, sustainable, and effective anti-aging strategies.

Annotated Checklist of the European Dacnusini and the Dapsilarthra genus group of the Alysiini (Hymenoptera: Braconidae, Alysiinae)

An annotated checklist of the European Dacnusini (426 species) and the Dapsilarthra genus group of the Alysiini (16 species) (Hymenoptera: Braconidae, Alysiinae) is provided. In addition to a species list with synonymy, further details are given of: (i) intrageneric groupings; (ii) a reference to each species’ treatment in the unindexed and multipart major revisions of Nixon & Griffith as well as the long keys of Tobias; (iii) a hypothesis about host range for the 60% of species which have been reared, and the evidence upon which it is based; (iv) whether a DNA barcode sequence is available (30% of species); (v) for species published after Griffiths’ revision a reference to similar species; (vi) any further relevant notes. One new synonym is established: Chorebus luzulae Griffiths syn. nov. is synonymised with Chorebus aphantus Marshall. Mesocrina Förster is excluded from the Dapsilartha genus group and whether Grandia Goidanich and Lodbrokia Hedqvist are in the Dacnusini is considered uncertain.

To pool or not to pool: Pooled metabarcoding does not affect estimates of prey diversity in spider gut content analysis

Abstract Gut content metabarcoding has provided important insights into the food web ecology of spiders, the most dominant terrestrial arthropod predators. In small invertebrates, like spiders, gut content analysis is often performed on whole body DNA extracts of individual predators, from which prey sequences are selectively enriched and sequenced. Since many spider species are generalist predators, large numbers of samples comprising individual spider specimens must be analysed to recover an exhaustive image of a spider species' prey spectrum, which is costly and time‐consuming. Pooled processing of bulk samples of multiple specimens has been suggested to reduce the necessary workload and cost while still recovering a representative estimate of the prey diversity. However, it is still unclear if pooling approaches lead to bias in recovering the prey spectrum and if the results are comparable with data from individually processed spiders. Here, we test the effects of metabarcoding pooled spider gut content on the recovered taxonomic diversity and composition of prey. Using a newly adapted primer pair, which efficiently enriches COI barcode sequences of diverse arthropod prey groups while suppressing spider amplification, we test if pooling leads to reduced taxonomic diversity or skewed estimates of prey composition. Our results show that pooling and individual processing recover highly correlated taxonomic diversity and composition of prey. The only exception are very rare prey items which were less well recovered by pooling. Our results support pooling as a cost‐effective and time‐efficient approach to recover the diet of generalist predators for population‐level studies of spider trophic interactions.