Standard DNA Sequencing Research Articles

First record of the family Callianopsidae (Decapoda: Axiidea) and a new species of Vulcanocalliax from the Hikurangi Margin off Aotearoa New Zealand, with a key to species of Callianopsidae

IntroductionThe Aotearoa New Zealand ghost shrimp of the infraorders Axiidea and Gebiidea have never been comprehensively reviewed, with recent work uncovering a diverse regional fauna representing eight of the 14 known families.MethodsUsing standard morphological and DNA sequencing tools, the family Callianopsidae is, for the first time, recorded off New Zealand, represented by a new species of Vulcanocalliax.ResultsThe new species was found near hydrocarbon seeps on the Hikurangi Margin, on the eastern New Zealand continental slope, and is only the second species now known in this genus. The single congener, V. arutyunovi, is only known from a mud volcano in the Gulf of Cádiz, off the Iberian Peninsula. Vulcanocalliax sp. nov. was formerly reported as an unnamed host of a new endemic New Zealand rhizocephalan barnacle Parthenopea australis and is here formally described as Vulcanocalliax beervana sp. nov.DiscussionThe new species differs, e.g., in the shape of the anterior carapace margin (convexly rounded and without elevated postantennal shoulder present in V. arutyunovi), the ocular peduncle having a convex anterolateral margin (compared to a straight margin), and the uropodal exopod has a dorsomedian ridge, lacking the elevated anterior portion that is distinct in V. arutyunovi. This brings the number of described New Zealand ghost shrimp species to 18. A key to all known Callianopsidae is provided.ZOOBANKLSID urn:lsid:zoobank.org:pub:4EDE6CE1-B644-4FFB-94CA-C8725A7549DD.

New specific primer matK and rbcL region for DNA barcode pitcher plant Nepenthes spathulata

Abstract. Utama MN, Etikawati N, Sugiyarto, Susilowati A. 2024. New specific primer matK and rbcL region for DNA barcode pitcher plant Nepenthes spathulata. Biodiversitas 25: 2515-2523. The matK and rbcL genes were proposed as the preferred plant barcoding loci by The Consortium for the Barcode of Life (CBOL). DNA barcoding efficiently identifies samples at the species level using short standard DNA sequences. The identification disclosure needs is an important basis given the confusion in systematics in Nepenthes spathulata. Therefore, this study aims to design matK and rbcL region-specific primers for DNA barcoding pitcher plants. The primary design uses Primer3Plus with a blueprint from the GenBank KDQ007081.1 for matK and MH346374.1 for rbcL. Following testing primary candidates with Oligo Analyzer and Primer-BLAST, the primers MkNs1, and RbNs4 were selected as the optimal criteria based on the values of ?G and primer specificity. Optimization of annealing temperature with PCR gradient shows that the temperature range 52-57°C produces a good band for both regions and corresponds to the matK and rbcL region with product sizes of 800-900 bp from seven samples, which could be observed from the gel electrophoresis. Sequence similarity using blast-N, the matK sequence has similarity to the N. spathulata matK gene of 99.64% and 99.63% for the rbcL gene of N. ventricosa x N. alata. This MkNs1 and RbNs4 primer can be used to discover the identity of N. spathulata and the Nepenthes genus.

Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine.

Multiomics require concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high-throughput DNA sequencing to include a controlled deamination step. Importantly, the rate of deamination of 5-methylcytosine is negligible and thus does not interfere with standard DNA sequencing and data processing. Thus, RIMS-seq2 libraries from whole- or targeted-genome sequencing show the same germline variation calling accuracy and sensitivity compared with standard DNA-seq. Additionally, regional methylation levels provide an accurate map of the human methylome.

Abstract 313: Benchmarking immune repertoire sequencing technologies

Abstract In the past decade, single cell gene expression technologies have transformed basic and translational research and are more frequently found in the toolbox of immunologists alongside the standard bulk B-cell receptor (BCR) sequencing and transcriptome profiling assays. While conventional bulk BCR assays capture the frequency of single chains, single-cell immune profiling reveals the frequency of paired heavy and light chains along with the cells transcriptional profile. Here we present the results of a single-blind benchmarking experiment conducted in collaboration with the U.S. Food and Drug Administration (FDA) that compares the performance of standard bulk DNA and RNA BCR repertoire sequencing assays, with the 10x Genomics Single Cell Immune Profiling solution. The FDA selected nine unambiguously monoclonal BCR-expressing cell lines to generate a reference mixture that was shared with each study participant, without revealing the input proportions. This reference cell line mixture was also diluted to 10% and 1% with B-cells sorted from peripheral blood mononuclear cells (PBMC) to further test the limits of clonotype detection. For each experiment, we targeted ~10,000 cells and in aggregate generated a paired gene expression and V(D)J dataset from ~100,000 cells. BCR clonotypes were detected in 78% of the captured cells across all samples profiled. Each of the 9 top unique clonotypes observed in our dataset corresponded to a cell line in the reference mixture, allowing us to decode the mixing percentages of each BCR-expression cell line. In addition, we were able to identify rare cell line clonotypes to as low as 0.01% in the dilution experiments. Furthermore, the multimodal nature of the single cell data also resolved the transcriptional profile of each cell line into its own cluster thus enabling independent verification of clonotype proportions. In summary, we have generated a resource for the immunology community to evaluate and compare data generated with different repertoire sequencing technologies. Citation Format: Shamoni Maheshwari, Carolyn Morrison, Sarah Taylor, BCR-SEQC consortium. Benchmarking immune repertoire sequencing technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 313.

DNA Barcoding of Locusta migratoria manilensis (Orthoptera: Acrididae) Reveals Insights into the Species and Subspecies Differentiation

Abstract Accurate identification and classification of insect species, especially those with significant economic and ecological implications, have historically presented challenges. Migratory locusts, Locusta migratoria manilensis (Meyen) (Orthoptera: Acrididae), are notorious for their destructive impact on crops. Traditional morphological methods often face limitations in distinguishing closely related species and require taxonomic expertise. However, the emergence of DNA barcoding as a powerful tool for species identification has revolutionized the field of entomology. DNA barcoding utilizes a standardized DNA sequence, a molecular barcode, which serves as a distinct genetic signature for rapid and accurate species identification. In this study, DNA barcoding techniques were employed to identify and differentiate the migratory locust subspecies manilensis, in both its solitary and gregarious forms, as well as to determine its phylogenetic relationship with other related species within the Acrididae family. GenBank reference sequences were used to identify the locusts at the molecular subspecies level. Although the COI marker did not exhibit significant differences between the solitary and migratory forms, it was valuable in resolving the identification of L. migratoria subspecies. This lack of significant differences may be attributed to limited genetic variation of COI at the subspecies level and substantial genetic similarities between the solitary and migratory forms, likely stemming from a recent common ancestor. Nonetheless, using COI remains beneficial for subspecies identification in migratory locusts.

Characterizing a standardized BioPart for BAG-specific expression in C. elegans.

Biological parts (BioParts) are modular and standardized DNA sequences that encode biological functions and contribute to the efficient biological engineering of complex systems. Here, we characterize a short BioPart (P flp-17 , 300 bp) for bright multicopy and single-copy BAG-specific expression starting from the gastrula stage in hermaphrodite and male C. elegans . We have generated standardized P flp-17 cloning vectors for BAG-specific gfp and mScarlet expression compatible with extra-chromosomal arrays and for single-copy transgene insertion. The short P flp-17 promoter is easy to generate by gene synthesis and has been incorporated into our online transgene design tool (www.wormbuilder.org/transgenebuilder ) .

Expanding the phylogeography and connectivity of Perkinsus species across North and Central America

AbstractAimParasites in the genus Perkinsus infect marine molluscs globally, with novel detections expanding and reshaping our knowledge of their biogeographic patterns and the factors influencing those patterns. Here, we aimed to characterize the phylogeography and genetic connectivity of Perkinsus spp. in bivalves across North and Central America, which included infection hot spots (e.g., the Gulf of Mexico, Chesapeake Bay) and areas where these parasites had not been previously reported (e.g., along the California coast).LocationAll coasts of North America and both coasts of Panama.MethodsUtilizing standard PCR and DNA sequencing, we genetically screened 933 bivalves from across North America for parasites in the genus Perkinsus, then combined this with 752 bivalves previously screened from Panama, which included 16 species from three different countries. Phylogenetic methods were used to confirm the identifications of all the bivalves collected and the Perkinsus spp. detected. We combined our data with publicly available sequence data for these parasites to create global haplotype networks to assess regional and continental genetic diversity and phylogeographic patterns.ResultsWe detected three species across North and Central America, including Perkinsus beihaiensis, P. chesapeaki, P. marinus, while P. olseni was only detected in Panama. We report for the first time P. chesapeaki was detected in Isognomon sp. from Mexico and P. beihaiensis detections in Ostrea lurida from California, USA. Additionally, our results indicate extremely low‐genetic diversity of P. beihaiensis, P. chesapeaki and P. marinus across continental and global spatial scales.Main conclusionsOur results add further evidence of recent or continuous long‐range dispersal and global connectivity for many haplotypes, suggesting that parasite dispersal through anthropogenic activities, such as maritime trade, likely contributes to these phylogeographic patterns.

Unexpectedly high prevalence of structural genomic variations in the Long QT Syndrome: evidence for broader clinical uptake

Abstract Background Long QT syndrome (LQTS) is an inherited channelopathy predisposing young and otherwise healthy individuals to ventricular arrhythmias causing syncope or sudden cardiac death. Point mutations in the KCNQ1, KCNH2, and SCN5A genes are the most common causes of LQTS, and up to 75% of patients can be genotyped by standard DNA sequencing. However, standard genetic testing fails to detect structural genomic rearrangements - including large deletions or duplications of DNA segments - known as copy number variants (CNVs). While previous studies suggested that CNVs account for less than 5% of genotype-negative cases(1-2), no recent data have been reported. Purpose The study aimed to determine the prevalence and spectrum of CNVs in LQTS susceptibility genes in unrelated patients who tested negative for point mutations in a panel of 16 genes associated with LQTS. Methods Next-generation sequencing (NGS) was used to screen CNVs in unrelated, clinically diagnosed LQTS patients without point mutations in 16 genes associated with LQTS. Loss of heterozygosity in LQTS-associated genes was used to assess the presence of CNVs using standard commercial software. The multiplex ligation-dependent probe amplification (MLPA) assay was used to confirm the CNVs identified by NGS. Results The cohort included 48 patients (63% women; mean age 31±19 years, range 3 years to 68 years) with definitive clinical diagnosis of LQTS (mean QTc 490±33 ms) according to the 2022 ESC guidelines(3). Overall, 7/48 patients (15%, 95% CI 6-28%) had CNVs. Six of the seven CNVs involved KCNQ1 in the heterozygous state, including three large deletions (exons 3-6, exons 12-15, and exon 16) and three duplications (exon 2, exons 3-6, and exons 3-15), whereas the remaining patient had a complete KCNH2 deletion. MLPA confirmed all CNVs identified by NGS. Of note, no CNVs were identified in LQTS-related genes whose pathogenicity has been questioned. Conclusions Our study highlights the unexpectedly high prevalence of structural genomic rearrangements in the KCNQ1 and KCNH2 genes in LQTS patients. Routine screening for copy number variants in LQTS patients is critical for accurate diagnosis, risk stratification, and personalized treatment strategies.Figure 1

Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA

The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we devise an enzymatic strategy for inserting a singular, orthogonal xenonucleic acid (XNA) base pair into standard DNA sequences using 2′-deoxy-xenonucleoside triphosphates as substrates. Integrating this strategy with combinatorial oligos generated on a chip, we construct libraries containing single XNA bases for parameterizing kmer basecalling models for commercially available nanopore sequencing. These elementary steps are combined to synthesize and sequence DNA containing 12 letters – the upper limit of what is accessible within the electroneutral, canonical base pairing framework. By introducing low-barrier synthesis and sequencing strategies, this work overcomes previous obstacles paving the way for making expanded alphabets widely accessible.

A new species of minute litter bug of the genus Cryptostemma (Heteroptera: Dipsocoromorpha: Dipsocoridae) from Basse-Terre (Guadeloupe Islands)

Summary A new species of the genus Cryptostemma Herrich-Schaeffer, 1835, C. karukeraensis n. sp., was collected in several localities of the island of Basse-Terre (Guadeloupe), by prospecting the particular micro-habitats specific to the Dipsocoridae. Males and females (macropterous) and 5th instar nymphs are described and illustrated, in particular details of the pregenital abdomen, and the highly asymmetric and modified genitalia in the male. Among its American congeners, the new species shows morphological similarities with C. haywardi Wygodzinsky described from Argentina, but differs in male and female genital characters. The standard barcode DNA sequence (COI) is given for this new species, which is the third known dipsocoromorphan representative from Guadeloupe, after Ceratocombus bifenestratus Poppius, 1910 (Ceratocombidae) and Schizoptera elegans Poppius, 1910 (Schizopteridae).

Heavy Metals Influence on the Bacterial Community of Soils: A Review

The increasing rate of natural resource use leads to an increase in the anthropogenic load on the soil. As the result of industrial, metallurgical and mining activities, excessive amounts of heavy metals (HMs) enter the soil. In addition, they can be introduced with waste and drainage water from various enterprises. Accumulating in the soil, HMs can negatively affect the soil bacterial community, which is one of the main factors of its “health”. Molecular genetic methods based on shotgun sequencing or metabarcoding of standard DNA sequences (for example, the 16S rRNA gene for bacteria) are the modern ways to assess the bacterial diversity of soils. This review presents the results of modern studies on the effect of HMs on the soil bacterial communities, using metagenomic methods. Based on the analysis of publications over the past two decades, a generally negative effect of pollutants on the taxonomic composition and diversity of bacterial communities has been shown. The influence of factors modulating the toxicity of metals and metalloids was noted: the amount and composition of salts, soil pH, ecosystem type, rhizosphere presence and other soil properties. In this paper, promising directions of research are outlined.

DDQR (dynamic DNA QR coding): An efficient algorithm to represent DNA barcode sequences.

A DNA barcode is a short piece of standard DNA sequence used for species determination and discrimination. Representation of DNA barcodes is essential for DNA barcodes' applications in the transportation and recognition of biological materials. Previously, we have compared different strategies for representing the DNA barcodes. In the present study, we have developed a compression algorithm based on binary coding or Huffman coding scheme, followed by converting the binary digits into Base64 digits. The combination of this compression algorithm and the QR representation leads to the dynamic DNA QR coding algorithm (DDQR). We tested the DDQR algorithm on simulated data and real DNA barcode sequences from the commonly used plant and animal DNA barcode markers: rbcL, matK, trnH-psbA, ITS2, and COI. We compared the compression efficiency of DDQR and another state-of-the-art DNA compression algorithm GeCo3 for sequences with various base compositions and lengths. We found that DDQR had a higher compression rate than GeCo3 for DNA sequences shorter than 800 bp, which is the typical size range for DNA barcodes. We also upgraded a web server (http://www.1kmpg.cn/ddqr) that provides three functions: retrieval of DNA barcode sequences, encoding DNA barcode sequences to DDQR codes, and decoding DDQR codes to DNA barcode sequences. The DDQR algorithm and the webserver will be invaluable to applying DNA barcode technology in the food and traditional medicine industries.

RCCC_Pred: A Novel Method for Sequence-Based Identification of Renal Clear Cell Carcinoma Genes through DNA Mutations and a Blend of Features.

To save lives from cancer, it is very crucial to diagnose it at its early stages. One solution to early diagnosis lies in the identification of the cancer driver genes and their mutations. Such diagnostics can substantially minimize the mortality rate of this deadly disease. However, concurrently, the identification of cancer driver gene mutation through experimental mechanisms could be an expensive, slow, and laborious job. The advancement of computational strategies that could help in the early prediction of cancer growth effectively and accurately is thus highly needed towards early diagnoses and a decrease in the mortality rates due to this disease. Herein, we aim to predict clear cell renal carcinoma (RCCC) at the level of the genes, using the genomic sequences. The dataset was taken from IntOgen Cancer Mutations Browser and all genes' standard DNA sequences were taken from the NCBI database. Using cancer-associated information of mutation from INTOGEN, the benchmark dataset was generated by creating the mutations in original sequences. After extensive feature extraction, the dataset was used to train ANN+ Hist Gradient boosting that could perform the classification of RCCC genes, other cancer-associated genes, and non-cancerous/unknown (non-tumor driver) genes. Through an independent dataset test, the accuracy observed was 83%, whereas the 10-fold cross-validation and Jackknife validation yielded 98% and 100% accurate results, respectively. The proposed predictor RCCC_Pred is able to identify RCCC genes with high accuracy and efficiency and can help scientists/researchers easily predict and diagnose cancer at its early stages.

Integrative taxonomy helps to revise systematics and questions the purported cosmopolitan nature of the type species within the genus Diaforobiotus (Eutardigrada: Richtersiusidae)

Recent advances in tardigrade taxonomy have been greatly enhanced by the redescriptions of the type species for particular taxa or species groups. De novo characterisation of these key taxa now allows to describe tardigrade species diversity with improved precision and at higher rate, increasing the momentum towards resolving the taxonomic impediment in these micro-invertebrates. Since its description, Diaforobiotus islandicus (Richters, 1904) has been reported from many distinct localities around the world. This suggested, perhaps falsely, a cosmopolitan nature of the species. However, potential erroneous assignment of newly found populations to this species could be a result of the very general and superficial original description. In order to properly recognise and name species diversity within the genus, I provide here an integrative redescription of the type species (D. islandicus) with a neotype designation, a description of a new species, Diaforbiotus svalbardicus sp. nov, and dichotomous key for the genus. Both descriptions are based on detailed morphological and morphometric data associated with standard DNA sequences of four genetic markers (18S rRNA, 28S rRNA, ITS-2, and COI). The genus composition and diagnosis amendments of the family Richtersiuside are also discussed. The presented study constitutes a starting point for further systematic studies on the genus Diaforobiotus and new taxa discoveries.

The preliminary DNA barcoding dataset of fishes from Chinese coastal waters

DNA barcoding is a molecular method for species identification based on a short standardized DNA sequence derived from the 5’ region of the cytochrome c oxidase subunit I (COI) gene, which is widely applied in marine fish identification. In this study, DNA barcodes were generated for 1,671 specimens representing 309 morphological marine fish species belonging to 223 genera and covering 101 families from Chinese coastal waters. The mean Kimura 2-parameter (K2P) genetic distances within species, genera, families, and orders were 0.20%, 20.94%, 24.45%, and 25.34%, respectively. As expected, the mean interspecific distance was higher than the mean intraspecific distance. Phylogenetic trees based on Bayesian inference (BI) and neighbor-joining (NJ) methods agreed with the taxonomic classifications. A total of 306 operational taxonomic units (OTUs) were reidentified via automatic barcode gap discovery (ABGD) analysis. An estimated nucleotide substitution matrices showed a transversion bias compared to transitional mutation. The amino acid variations of the barcoding region were also examined, and 13 amino acid sites exhibited high variability. Overall, our findings confirmed that DNA barcoding is a highly reliable mean of identifying fish species in Chinese coastal waters, and that the barcode library established here would facilitate the monitoring and management of the marine fishery resources in China.

A REVIEW METHOD FOR IDENTIFICATION OF RARE AND ENDANGERED PLANTS THROUGH DNA BARCODING

DNA barcoding is a new concept. It has been developed for providing fast, precise and automatable species identification which uses standardized DNA sequences as tags. DNA barcoding can provide the taxonomists; conservationists. The early goal of the DNA barcoding process is to build online libraries of barcode sequences for all known species that can serve as a standard to which DNA barcodes of any identified or unidentified specimens can be matched. This can improve several inherent problems related with traditional taxonomic identification, based on morphological characters, such as incorrect identification of species due to phenotypic plasticity and genotypic variability of the characters, such as incorrect identification of species due to phenotypic plasticity and genotypic variability of the characters, overlooking cryptic taxa, difficulty in finding reliable characters due to long maturity periods (CBOL Plant Working Group, 2009). It is particularly of much use in areas where species identification with morphological characters is not practicable due to widespread damage or delayed expression. It should be enduring in mind that DNA barcoding is not an alternative to taxonomy, and it cannot replace taxonomy as such, but is a useful tool that creates information on unknown taxa. In this paper, methods of the process of selecting and redefining barcodes for plants evaluation of the factors which manipulate the discriminatory power of the advance with some early applications of DNA barcoding are discussed and then added the authors’ for their views and recommendations.

Mammo Screen Artificial Intelligence (AI) Tool Improves Diagnostic Performance of Radiologists in Detecting Breast Cancer

The abundance of T cells helps predict the patient's response to immunotherapy, so researchers hope this new method could provide specific and more effective treatments for cancer. Scientists analyzed DNA sequencing data from patients 'cancerous tumors to see if they could detect T cell deficiency. DNA sequencing is often performed on cancerous patients' tumors. It is done to classify them and understand how the cancer progresses. Estimation of immune cells, which are important for cancer control, affects patient survival and guides treatment. Our goal was to be able to develop a new method for annotating immune cells directly from DNA sequences without the need for further data. DNA sequencing allows scientists to see the history and evolution of tumors. In this study, scientists developed a way to calculate the historical levels of T cells; By reassembling or modifying them, they are provided with tools that allow them to detect attackers. In particular, the scientists found a "signal" for the loss of TREC cell division, and by recording this, they were able to accurately estimate the number of T cells in the tumor. In recent years, screening inhibitors (IPCs), a type of immunotherapy, have emerged as a revolutionary treatment for many types of cancer. ICPs work by blocking proteins called checkpoints made by T lymphocytes. These checkpoints prevent strong immune responses, as they can sometimes prevent T lymphocytes from killing cancer cells. When these checkpoints are closed, T cells are better able to kill cancer cells. One of the biomarkers showed that the potential success of immunotherapy was predicted by the number of T cells available, and that the more T cells available to IPCs, the more cancer cells were killed. It provides the patient's response to treatment without the need for additional data. In fact, the process we have developed can go beyond the standard DNA sequence at no extra time or cost. Our tools also make it possible to study the immune system more than just cancer. Keywords: Cancer; Cells; Tissues; Tumors; Prevention; Prognosis; Diagnosis; Imaging; Screening; Treatment; Management

Ultra-accurate microbial amplicon sequencing with synthetic long reads

BackgroundOut of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge.MethodsHere, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads.ResultsLoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens.ConclusionsThe order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.4PHa9SHy5rS1yXExpPUL7_Video abstract.

Antibiotic Effects of selected leaf extracts and molecular profile of Klebsiella pneumoniae and Enterobacter bugandensis strain (AdM2)

Only healthy surface tissues of healthy animals that are constantly in contact with the environment can easily be colonized by various microbial species, including pathogenic ones. However, these microorganisms are usually the cause of opportunistic infections in animals. Two plants; Vernonia amygdalina (taxonomic ID number 112605) and Ocimum gratissimum (taxonomic ID number 112606) were identified at the Nigerian Federal Research Institute in Ibadan and subsequently concentrated with methanol, water and n-hexane. Twelve pre-slaughter healthy cattle in the same ratio of male to female were selected for this study. Samples from the mouth, skin, nose, vaginal and gastrointestinal tract were collected. Susceptibility patterns for antimicrobial agents were performed using disk-diffusion agar method. All isolates were subjected to bacteriological and molecular identification (PCR and sequencing techniques). Preliminary identification was carried out based on standard bacteriological, microscopic identification, biochemical criteria, while PCR and DNA sequencing techniques were used for molecular identification and analysis. Basic local alignment sequence tools (BLAST) were used to compare retrieved bacterial sequences to a set of previously published strains in the database. The morphological and biochemical profiles of six out of eight normal flora from different sites were Gram-negative. All bacterial cultures were oxidase-positive, including 7 catalase-positive cultures. The majority of isolated bacteria were moderately sensitive to all the extracts tested but resistant to Amoxylin. Noticeable amplification of 16S rRNA genes from Klebsiella pneumoniae strain EMB and Enterobacter bugandensis strain AdM2 was obtained. The amplification of the resistant genes of the two isolates was approximately 1,500 base pairs; however, some of the suspected virulence and antibiotic genes were within this base pair range.

A new redescription of Richtersius coronifer, supported by transcriptome, provides resources for describing concealed species diversity within the monotypic genus Richtersius (Eutardigrada)

Richtersius coronifer, the nominal species for the family Richtersiidae and a popular laboratory model, exemplifies a common problem in modern tardigrade taxonomy. Despite undeniable progress in the field, many old and incomplete descriptions of taxa hinder both species delimitation and the estimation of species diversity and distribution. Although for over a century this species has been recorded throughout the world, recent research indicates that records to date are likely to represent a species complex rather than a single cosmopolitan species. However, in order to recognise and name species diversity within the complex, an integrative redescription of the nominal species is first needed. Here, we describe an R. coronifer population collected from Spitsbergen, i.e., one of the two localities mentioned in the original description, with detailed morphological and morphometric data associated with standard DNA sequences of four standard genetic markers (18S rRNA, 28S rRNA, ITS-2, and COI) and supported by transcriptome sequencing. We propose replacement of the neotype designated in 1981 by Maucci and Ramazzotti, as it is impossible to verify whether the existing neotype is conspecific with specimens studied by Richters in 1903 and 1904. Finally, using newly obtained cytochrome c oxidase subunit I (COI) sequences of populations from Spitsbergen, Italy, Poland, and Greece together with sequences deposited in GenBank (China, Greenland, Italy, Mongolia), we performed genetic species delimitation, which indicated seven distinct potential species within the genus Richtersius, in addition to the nominal taxon. This study marks a starting point for further research on the taxonomy of and species diversity within the genus. Moreover, this work has the potential to be the first tardigrade redescription to provide both genetic barcodes and a transcriptome of the species in question.