Molecular Barcoding Research Articles

DNA barcoding of five economically important freshwater fish species from the Nile River, Sudan

The study was conducted to barcode five freshwater fish species obtained from Nile River tributaries (the Atbara River and the Blue Nile), in March 2019, using analysis of partial CO1 gene sequences. Fish samples were identified morphologically as Nile perch Lates niloticus, Nile labeo Labeo vulgaris, North African catfish Clarias gariepinus, Cornish jack Mormyrops anguilloides and bebe mormyrid Hyperopisus bebe. Following DNA extraction, universal primers were used for amplification of the CO1 barcode region. The PCR products were sequenced and used for molecular identification. Each sequence was matched to corresponding GenBank sequences using the BLAST technique. Sequences of all specimens were aligned along with the GenBank published sequences and polymorphic sites were identified. The analysed sequences were uploaded to GenBank under the accession numbers OL804282– OL804286. The analysed partial CO1 barcode gene showed a readable length of ∼548–669 base pairs. Sequence comparison of CO1 gene sequences with sequences published in GenBank revealed 99.27–100% identities at the species level. Therefore, the CO1 barcodes accurately identified all the studied freshwater fishes. This is the first molecular DNA barcode characterisation of freshwater fish species found in Sudan. The fish CO1 gene sequences revealed in this study will serve as barcode references for future characterisations of Nile River fishes.

Cryptic species in time and space: an assessment of cryptic diversity within eight nominal species of Hydrozoa (Cnidaria).

Sampling in multiple localities, coupled with molecular barcoding, has shown that nominal species with wide geographical distribution often harbour local cryptic species in allopatry. Cryptic species in sympatry, however, can be easily missed if they have different seasonality, because they can be identified only through long-term frequent sampling (i.e. sampling through time of the same species in the same location). This is especially true in planktonic invertebrates that exhibit strong seasonality. By integrating mitochondrial 16S sequences of eight species of Hydrozoa (Cnidaria) collected weekly for a year in one Gulf of Mexico region, with sequences gathered globally, we investigate the presence of cryptic species within a temporal gradient (regionally) and on a spatial (worldwide) scale. We find that eight species of Hydrozoa are composed of 28 cryptic species, with 16 of them appearing in sympatry but with non-overlapping seasonality. The high number of sympatric cryptic species could only be discovered through extensive and prolonged regional sampling efforts. The bi-dimensional cryptic diversity (in time and space) highlighted in this study is essential for understanding processes of evolution, biogeography dispersal in the sea, and for more realistic biodiversity assessments.

Bryophyte molecular barcoding records. 8

Bryophyte molecular barcoding records. 8

First Report of Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst Nematode) Infecting Corn (Zea mays) in Spain.

Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst nematode) is an important disease of corn in several areas of the world, including India, Nepal, Pakistan, Egypt, USA, Greece and Portugal (Subbotin et al., 2010). It is a sedentary semi-endoparasite feeding on corn roots and other Poaceae plants and has been associated with significant yield losses in corn (Subbotin et al., 2010). During autumn 2022 a plant-parasitic nematode survey performed in corn at central-western area of Spain (Talavera de la Reina, Toledo), revealed a commercial field with stunted plants. Nematodes were extracted from soil by centrifugal-flotation method (Coolen, 1979). Corn roots inspection detected infections by immature and mature cysts, and soil revealed also mature live cysts and second-stage juveniles (J2s) with a population density of 1010 eggs and J2s/500 cm3 soil (including eggs from cysts). J2s and cysts were processed to pure glycerine using De Grisse's (1969) method. DNA was isolated from single live fresh J2s specimens for amplifying and sequencing of cytochrome c oxidase subunit II (COII) mitochondrial region using the primer pair species-specific H.Gly-COIIF_inFOR/P116F-1R (Riepsamen et al., 2011); D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A/D3B primers (De Ley et al. 1999); internal transcribed spacer (ITS) region using primers TW81/AB28 (Subbotin et al., 2001); and cytochrome c oxidase subunit 1 (COI) gene was amplified using the primers JB3/JB5 (Bowles et al., 1992). Brown cysts were lemon-shaped with a protruding vulval cone with fenestra ambifenestrate, bullae prominent below underbridge and characteristically arranged in finger-like bullae (Fig. 1). J2 with slightly offset lip region (3-5 annuli), stylet strong with rounded stylet knobs, lateral field with four lines, and tail short and tapering conically. Measurements of cysts (n=10) included body length 559 (432-688) µm, body width 450 (340-522) µm, fenestral length 40 (36-43) µm, semifenestral width 19 (17-21) µm, and vulval slit 40 (35-44) µm. J2 measurements (n=10) included body length 477 (420-536) µm, stylet length 21 (20-22) µm, tail length 51 (47-56) µm, and tail hyaline region 23 (20-26) µm. Morphology and morphometrics of cysts and J2, fit with original description and others from several countries (Subbotin et al., 2010). Two J2s individuals were sequenced for COII region (OQ509010-OQ509011) showing 97.1-98.1% similarity with H. zeae from USA (HM462012). Six almost identical 28S rRNA sequences from J2s (OQ449649-OQ449654) were 99.2-99.4% similar to 28S rRNA sequences of H. zeae from Greece, Afghanistan and USA (GU145612, JN583885, DQ328695). Four identical ITS DNA fragments from J2s (OQ449655-OQ449658) were 97.0-97.8% similar to ITS sequences of H. zeae from Greece, and China (GU145616, MW785771, OP692770). Finally, six COI sequences of 400 bp obtained for J2s (OQ449699-OQ449704) were under 87% similarity to several COI sequences of Heterodera spp. in NCBI, being a new molecular barcoding for identifying this species. On the basis of these results, the cyst nematodes isolated from the corn plants from the central-western area of Spain (Talavera de la Reina, Toledo) were confirmed as H. zeae and up to our knowledge it is the first report in Spain. This is a well-known pest of corn, causing important losses in this crop (Subbotin et al., 2010) and it was previously regulated as a quarantine nematode in the Mediterranean region (EPPO).

Enchytraeids in imported soil and organic deposits in Pyramiden, an abandoned mining town on Spitsbergen in the High Arctic

In Arctic soils, potworms (Clitellata: Enchytraeidae) are a particularly important decomposer group. There are relatively many faunistic records from the Svalbard archipelago, in particular its main island Spitsbergen, but few ecological studies on enchytraeids. With climate change and increasing tourism, the introduction and subsequent spreading of exotic species might pose a threat to native biotic communities. In Pyramiden, a Russian (formerly Soviet) coal mining settlement on Spitsbergen abandoned in 1998, Chernozem soil from southern Russia or Ukraine had been imported in 1983 to create lawns. Today the long central lawn presents an environmental gradient: on one end rather shallow humus-rich soil is fertilized by a sea bird colony on an adjacent building, the central part has a rich organic horizon topping humus-rich mineral soil, whereas on the other end humus-rich soil is covered by deposited mineral soil. In August 2018, we sampled this gradient to study the enchytraeid assemblage. Additional samples were also taken within frames with a vigorous grass turf in front of residential houses and in a vegetated heap of decomposing organic material (mostly woodchips). The objectives were to see (1) if and how the differences between habitats would be reflected by changes of the enchytraeid assemblages and (2) if there were any potentially introduced species. Almost 3700 specimens were identified and 9 species were recorded, which is high for habitats of the High Arctic. One species, Enchytraeus buchholzi s.l., had been recorded before in the archipelago only once, also in imported soil. This small-bodied euryoecious r-strategist is abundant in agricultural and other disturbed soils. Molecular barcoding and comparison with sequences from Central Europe showed close resemblance to several specimens, which makes an introduction with the transferred Chernozem highly probable. Most enchytraeids were concentrated in the upper 3 cm of soil (even if mineral), only in the organic deposits vertical distribution was rather even down to 12 cm depth. Numbers in the organic deposits were exceedingly high (ca 417,000 individuals/m2) and E. buchholzi s.l. represented ca 90 % of all specimens. Ordination (NMDS) showed all habitats to host rather distinct assemblages. Spearman's rank correlation showed significant positive correlation of species richness with conductivity and total nitrogen and of abundance with phosphorous and potassium contents in aqueous solution.

First insight into molecular diversity and DNA barcode library of epikarst-dwelling invertebrates in the Western Carpathians

DNA barcoding represents a handy tool for species identification. In addition, it serves as a complementary approach that improves the characterisation of evolutionary lineages and facilitates the detection of potentially undescribed and cryptic species. Based on the case study in the Western Carpathians, which belong to the Carpathian biodiversity hotspot, we have compiled the first DNA barcode reference library for molecular identification of invertebrates associated with epikarst, a unique, yet understudied, shallow subterranean aquatic habitat that extends at the interface between the soil and carbonate rocks. We analysed invertebrates collected in 2019-2020 from epikarst water that continuously seeps into four caves of the Demänovský Cave System in northern Slovakia. The standard barcode marker of the mitochondrial COI gene was amplified in more than 920 individuals of aquatic, semi-aquatic, and terrestrial invertebrates. The final data set consisted of 784 barcode sequences representing 36 morphospecies, the majority (98.3%) belonging to Arthropoda. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 60 Barcode Index Numbers (BINs), of which 43 BINs were new to BOLD, representing mostly typical subterranean species. Almost 20% of the morphospecies displayed high intraspecific variation (>2.2%), suggesting the need for further investigation to assess potential taxonomic problems or cryptic diversity. Our results also indicated the existence of several yet undescribed invertebrate species and possible heteroplasmy or COI numts in the collembolan Megalothorax sp. (incertus species group). The resulting DNA barcode library represents a significant advance not only in the characterisation of epikarst biodiversity but also in the understanding of subterranean biodiversity in general, paving the way for future complex evolutionary and biogeographical studies.

Bacterial Species Associate on the Body Surface of Musca domestica L from Various Habitats based on 16S rRNA Sequencing

This study aims to identify bacteria isolated from the body surface of house flies from various habitats using 16S rRNA molecular barcodes. Houseflies were isolated from forests, hospitals, traditional markets, modern markets and landfills. 25 house flies isolated in each habitat. House flies were preserved in sterile bags. Bacterial isolation was carried out using nutrient agar media in 100 mm Petri dishes. The isolates obtained were pure cultured until a single isolate was obtained. Single isolates were extracted using Geneaid’s Presto TM Mini gDNA Bacteria Kit. The extracted bacterial total DNA was used as a template for amplification using primer 16s rRNA gene by PCR method. Nucleotide sequencing uses Singapore’s First BASE sequencing service. The results showed that single-house fly isolates from the Fish Auction (P.L.) showed a 99.11% similarity with Sphingobacterium faecium [CP094931.1]. Traditional market bacterial isolates (P.T.) showed 97% similarity with Pseudochrobactrum sp. XF203. Hospital bacterial isolates (R.S.) showed 99.11% similarity with S. faecium [CP094931.1]. Bacterial isolates from residential areas (PM) showed 99% similarity with Brucella abortus RB51-AHVLA. Bacterial isolates from the forest (H.T.) showed 94% similarity with Bacillus paralicheniformis [CP043501.1]. There are associated bacteria that are used as biotechnology agents. Exploration of bacteria and even microbes associated with M. domestica is still extensive to be studied in the future.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Distinct neuroinflammatory signatures exist across genetic and sporadic amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia, referred to as ALS-frontotemporal spectrum disorder (ALS-FTSD). For mutations associated with ALS-FTSD, such as the C9orf72 hexanucleotide repeat expansion, the molecular factors associated with heterogeneity along this spectrum require further characterization. Here, using a targeted NanoString molecular barcoding approach, we interrogate neuroinflammatory dysregulation and heterogeneity at the level of gene expression in post-mortem motor cortex tissue from a cohort of clinically heterogeneous C9-ALS-FTSD cases. We identified 20 dysregulated genes in C9-ALS-FTSD, with enrichment of microglial and inflammatory response gene sets. Two genes with significant correlations to available clinical metrics were selected for validation: FKBP5, a correlate of cognitive function, and brain-derived neurotrophic factor (BDNF), a correlate of disease duration. FKBP5 and its signalling partner, NF-κB, appeared to have a cell type-specific staining distribution, with activated (i.e. nuclear) NF-κB immunoreactivity in C9-ALS-FTSD. Expression of BDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™ in situ hybridization. Our analyses also revealed two distinct neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. We compared NPS between C9-ALS-FTSD cases and those from sporadic ALS and SOD1-ALS cohorts and identified NPS1 and NPS2 across all cohorts. Moreover, a subset of NPS was also able to separate publicly available RNA sequencing data from independent C9-ALS and sporadic ALS cohorts into two inflammatory subgroups. Importantly, NPS subgroups did not clearly segregate with available demographic, genetic, clinical or pathological features, highlighting the value of molecular stratification in clinical trials for inflammatory subgroup identification. Our findings thus underscore the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within ALS-FTSD cohorts.

A versatile CRISPR-based system for lineage tracing in living plants.

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Intragenomic diversity of the V9 hypervariable domain in eukaryotes has little effect on metabarcoding

Metabarcoding revolutionized our understanding of diversity and ecology of microorganisms in different habitats. However, it is also associated with several inherent biases, one of which is associated with intragenomic diversity of a molecular barcode. Here, we compare intragenomic variability of the V9 region of the 18S rRNA gene in 19 eukaryotic phyla abundant in marine plankton. The level of intragenomic variability is comparable across all the phyla, and in most genomes and transcriptomes one V9 sequence and one OTU is predominant. However, most of the variability observed at the barcode level is probably caused by sequencing errors and is mitigated by using a denoising tool, DADA2. The SWARM algorithm commonly used in metabarcoding studies is not optimal for collapsing genuine and erroneous sequences into a single OTU, leading to anoverestimation of diversity in metabarcoding data. For an unknown reason,SWARM inflates diversity of eupelagonemids more than that of other eukaryotes.

Morphological and Molecular Barcoding Characterization of Four Invasive Species of Solanaceae in the Southern Region of Saudi Arabia

Invasive plants are the second greatest threat to plant diversity, after habitat loss due to human activities. Plant diversity hotspots and islands, as well as farmlands, woodlands, forests, grasslands, and populated areas, are all threatened by invasive species. Over the centuries, and especially in the last few decades, trade and travel have increased dramatically, hastening the introduction of alien species. Although comprehensive sampling is necessary for DNA barcoding, it is rarely done due to a lack of materials. In practice, only a few species from each genus must be identified, rather than all of them. In many of Saudi Arabia's most problematic habitats in the southwestern region, where the presence of invasive species was significant. As a result, in the current application of DNA barcodes, identifying a given species using a limited sample is important. In the case of incomplete sampling, DNA barcodes can provide reliable specific-species discrimination. For that purpose, the current study was designed to identify the four invasive species in the southern part of Saudi Arabia using morphological and 4 molecular barcoding (ITS, PsbA, rbcla, matk,) markers were used for the identification and characterization of invasive species. The results suggested identifying the 4 invasive Solanaceae plants species in the southwestern region. A phylogenetic tree was also constructed from the identified four species along with reference sequences of the blast results from the NCBI confirmed the four Solanaceae species i.e., Datura innoxia, Datura stramonium, Nicotiana glauca, and Withania somnifera, recorded invasive in the southern part of Saudi Arabia. Except for the matK tree, all the trees clustered individuals of the same species together and separately from the other species, with strong bootstrap support indicating that they were more closely related species.

Barcoding coffee grounds - Exploring pteropod gastropod biodiversity with dregs in collection jars

Despite their cosmopolitan occurrence and massive plankton sampling during expeditions, the genetic diversity within Pteropoda Cuvier, 1804 is still largely unexplored. In this study we present a next-generation environmental barcoding approach to zooplankton bulk samples, which were collected during the circumglobal 2010 Malaspina expedition to evaluate pteropod diversity. We introduce a technique that avoids destructive procedures and leaves material intact for further morphological investigations. We extracted DNA out of the dregs (organic material such as mucus or body parts) of 27 sample containers for molecular barcoding (average 100-260 bp of COI). We were able to identify 7128 operational taxonomic units corresponding to the species composition contained in the examined samples. Among them were three species of thecosome pteropods, Creseis acicula, Creseis virgula and Cavolinia inflexa, which are discussed with respect to their taxonomy and their geographic distribution. Unidentified gymnosomes were also present in our samples from warmer regions in oceanic waters of the southern Indian Ocean. To facilitate identification of species, it is beneficial to create a better database of pteropod COI barcodes. Furthermore, gathering environmental barcoding data on a broad global scale will help to better understand species abundance and distribution of pteropods in the world’s oceans, and potentially those of other planktonic organisms.

Marine biodiversity discovery: the metrics of new species descriptions

Based on the World Register of Marine Species (WoRMS), there are currently c. 242,000 known valid marine species living in the world's oceans and marine biota continue to be discovered and named steadily at a current average of 2,332 new species per year. The “average” newly described marine species is a benthic crustacean, annelid, or mollusc between 2 and 10 mm in size, living in the tropics at depths of 0–60 m, and represented in the description by 7–19 specimens. It is described after a shelf life of 13.5 years in an article with two to three authors in a journal with an IF <1, published by an academic institution or society or a small commercial publisher. It is highly likely that the description is not accompanied by molecular data and that its authors do not work in an institution in a region of the world where the new species comes from. At the current pace of discovery and characterization, it will take several hundred years to describe the remaining 1–2 million unknown marine species. With increased facilitation of access to literature, marine taxonomy will increasingly rely on retired professionals and citizen scientists. The barriers to new marine species descriptions are in part technological (access to habitats that are difficult to sample) and educational (training to generate and use molecular barcodes), but mostly institutional (funding of taxonomic work) and regulatory (restrictions imposed by access and benefit sharing legislation).

The microbial composition of larval airways from Drosophila melanogaster differ between specimens from laboratory and natural habitats

BackgroundThe fruit fly Drosophila melanogaster lives in natural habitats and has also long been used as a model organism in biological research. In this study, we used a molecular barcoding approach to analyse the airways microbiome of larvae of D. melanogaster, which were obtained from eggs of flies of the laboratory strain w1118 and from immune deficient flies (NF-kB-K), and from wild-caught flies. To assess intergenerational transmission of microbes, all eggs were incubated under the same semi-sterile conditions.ResultsThe airway microbiome of larvae from both lab-strains was dominated by the two families Acetobacteraceae and Lactobacillaceae, while larvae from wild-caught flies were dominated by Lactobacillaceae, Anaplasmataceae and Leuconostocaceae. Barcodes linked to Anaplasmataceae could be further assigned to Wolbachia sp., which is a widespread intracellular pathogen in arthropods. For Leuconostoceae, the most abundant reads were assigned to Weissella sp. Both Wolbachia and Weissella affect the development of the insects. Finally, a relative high abundance of Serratia sp. was found in larvae from immune deficient relish−/− compared to w1118 and wild-caught fly airways.ConclusionsOur results show for the first time that larvae from D. melanogaster harbor an airway microbiome, which is of low complexity and strongly influenced by the environmental conditions and to a lesser extent by the immune status. Furthermore, our data indicate an intergenerational transmission of the microbiome as shaped by the environment.

Morphological and Molecular Identification of Jujube Fruit Fly Carpomya incompleta (Becker, 1903) (Diptera: Tephritidae) in southeast Iraq

An accurate identification of an insect is essential for the effective pest management program. However, due to the restrictions of phenotypic identification, molecular process (e.g. DNA barcoding) is used to overcome some of these limitations. The present paper was aimed to identify the Jujube fruit fly by morphological characters of the adults and molecular DNA barcoding of the larval stage in southeast Iraq. The adults was morphologically identified as Carpomyia incompleta (Becker, 1903) (Diptera: Tephritidae). Results of the molecular method revealed that the mitochondrial COI – COII marker was successfully identified immature stage of the species. The sequence analysis results identified the specimens (AHL1 and AHL2) as C. incompleta. Sequences of the specimens were recorded in The US National Center for Biotechnology Information (NCBI) under accession numbers (ON045002.1 and ON045003.1, respectively. These results were consistent with the phenotypic identification of the Jujube fruit flies.

Molecular Barcoding: A Tool to Guarantee Correct Seafood Labelling and Quality and Preserve the Conservation of Endangered Species.

The recent increase in international fish trade leads to the need for improving the traceability of fishery products. In relation to this, consistent monitoring of the production chain focusing on technological developments, handling, processing and distribution via global networks is necessary. Molecular barcoding has therefore been suggested as the gold standard in seafood species traceability and labelling. This review describes the DNA barcoding methodology for preventing food fraud and adulteration in fish. In particular, attention has been focused on the application of molecular techniques to determine the identity and authenticity of fish products, to discriminate the presence of different species in processed seafood and to characterize raw materials undergoing food industry processes. In this regard, we herein present a large number of studies performed in different countries, showing the most reliable DNA barcodes for species identification based on both mitochondrial (COI, cytb, 16S rDNA and 12S rDNA) and nuclear genes. Results are discussed considering the advantages and disadvantages of the different techniques in relation to different scientific issues. Special regard has been dedicated to a dual approach referring to both the consumer's health and the conservation of threatened species, with a special focus on the feasibility of the different genetic and genomic approaches in relation to both scientific objectives and permissible costs to obtain reliable traceability.

New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution.

Wood-inhabiting fungi (WIF), such as polypores, are extremely species-rich and play vital roles in the functioning of forest ecosystems as decomposers. Despite the importance of polypores, our knowledge of the diversity and distribution of these fungi is still poor in general and especially for West Africa. To advance our knowledge we here summarise results from field collections between 2017 and 2021 and present (i) a taxonomic overview, (ii) phylogenetic placements and (iii) an illustrated catalogue of wood-inhabiting polypore fungi with colour pictures. During the field sampling campaigns, we collected 647 specimens. Based on morphological characteristics and molecular barcode data, 76 polypore species belonging to six orders, 15 families and 39 genera were identified. Of the 76 species, 30 are new to the West Africa, 69 new to Benin, and two new combinations Fuscoporia beninensis and Megasporia minuta are proposed. With this summary, we provide new data for further research. Citation: Olou BA, Langer E, Ryvarden L, Krah F-S, Hounwanou GB, Piepenbring M, Yorou NS (2023). New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution. Fungal Systematics and Evolution 11: 11-42. doi: 10.3114/fuse.2023.11.02.

METB-13. A SINGLE-CELL GENETIC IN VIVO LINEAGE-TRACING PLATFORM FOR MEDULLOBLASTOMA

Abstract Comparative single-cell studies of group 3/4 medulloblastomas (MBs) and fetal brain have identified a common hierarchy of glutamatergic-lineage cell types and an apparent cell-of-origin in the rhombic lip. An increased understanding of the genetic regulators of stem-cell maintenance and differentiation in MB would clearly be of therapeutic benefit. There is a significant need for lineage-tracing systems to model MB malignant transformation, progression, and response to therapy. We developed in vivo and in organoid genetic lineage-tracing systems with a single-cell readout. A high-complexity lentiviral library expressing heritable polyadenylated molecular barcodes was transduced into D283 and D425 patient-derived cell lines. Barcoded cells were injected into the brains of immunocompromised mice. Both preimplantation barcoded cultures and the resulting mature tumors were profiled by single-cell RNA-sequencing (scRNA-seq). This captured both endogenous RNA and barcode transcripts. While only minimal barcode clash was detected in the preimplantation cultures, we observed a clonal expansion of barcodes in mature tumors which aligned with phylogenetics analysis of expressed mutations in endogenous RNA. Methods for assessing lineage coupling between transcriptional clusters and inferring their lineage relationship were developed. Ongoing efforts to recover barcodes from tumor sections via spatial transcriptomics will be presented. These studies fill a gap in status quo models of MB which are needed to leverage recent findings derived from single-cell analysis of human tumors.

Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria

BackgroundHippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria.MethodsLouse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses.ResultsA total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida.ConclusionsMolecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.Graphical