The Cosmonaut Sea is one of the least accessed regions in the Southern Ocean, and our knowledge about the fish biodiversity in the region is sparse. In this study, we provided a description of demersal fish diversity in the Cosmonaut Sea by analysing cytochrome oxidase I (COI) barcodes of 98 fish samples that were hauled by trawling during the 37th and 38th Chinese National Antarctic Research Expedition (CHINARE) cruises. Twenty-four species representing 19 genera and 11 families, namely, Artedidraconidae, Bathydraconidae, Bathylagidae, Channichthyidae, Liparidae, Macrouridae, Muraenolepididae, Myctophidae, Nototheniidae, Paralepididae and Zoarcidae, were discriminated and identified, which were largely identical to local fish occurrence records and the general pattern of demersal fish communities at high Antarctic shelf areas. The validity of a barcoding gap failed to be detected and confirmed across all species due to the indicative signals of two potential cryptic species. Nevertheless, DNA barcoding still demonstrated to be a very efficient and sound method for the discrimination and classification of Antarctic fishes. In the future, various sampling strategies that cover all geographic sections and depth strata of the Cosmonaut Sea are encouraged to enhance our understanding of local fish communities, within which DNA barcoding can play an important role in either molecular taxonomy or the establishment of a dedicated local reference database for eDNA metabarcoding analyses.

Sheep and goat may become carriers of some zoonotic diseases. They are important livestock and experimental model animals for human beings. The fast and accurate identification of genetic materials originating from sheep and goat can prevent and inhibit the spread of some zoonotic diseases, monitor market product quality, and maintain the stability of animal husbandry and food industries. This study proposed a methodology for identifying sheep and goat common specific sites from a genome-wide perspective. A total of 150 specific sites were selected from three data sources, including the coding sequences of single copy genes from nine species (sheep, goat, cow, pig, dog, horse, human, mouse, and chicken), the dbSNPs for these species, and human 100-way alignment data. These 150 sites exhibited low intraspecific heterogeneity in the resequencing data of 1450 samples from five species (sheep, goat, cow, pig, and chicken) and high interspecific divergence in the human 100-way alignment data after quality control. The results were proven to be reliable at the data level. Using the process proposed in this study, specific sites of other species can be screened, and genome-level species identification can be performed using the screened sites.

OBJECTIVE: Urinary tract infections are the most common hospital-acquired infection, 80% are associated with catheterisation. Diagnostic methods may influence the reported identities of these pathogens, and phenotypic testing under laboratory conditions may not reflect infection phenotypes. This study aimed to evaluate the efficacy of diagnostic methods and whether medium composition alters phenotypes by characterizing catheter-associated urinary tract infection isolates from a UK hospital.
RESULTS: We compared five bacterial identification methods, including biochemical testing, MALDI biotyping, and genome sequencing, finding differences in genus or species level identifications. Antibiotic susceptibility comparisons between phenotypic assays and genomic predictions showed high agreement only in multidrug-resistant strains. To determine whether growth rate and biofilm formation were affected by medium composition, strains were grown in both planktonic and biofilm states. Low planktonic growth and significant biofilm formation were observed in artificial urine compared to rich laboratory media, underscoring the importance of assay design.
CONCLUSIONS: This study highlights the risks of relying on a single diagnostic method for species identification, advocating for whole-genome sequencing for accuracy. It emphasizes the continued importance of phenotypic methods in understanding antibiotic resistance in clinical settings and the need for characterization conditions that mirror those encountered by pathogens in the body.

Terrestrial orchids are a group of genetically understudied, yet culturally and economically important plants. The Orchidinae tribe contains many species that produce edible tubers that are used for the production of traditional delicacies collectively called \'salep\'. Overexploitation of wild orchids in the Eastern Mediterranean and Western Asia threatens to drive many of these species to extinction, but cost-effective tools for monitoring their trade are currently lacking. Here we present a custom bait kit for target enrichment and sequencing of 205 novel genetic markers that are tailored to phylogenomic applications in Orchidinae s.l. A subset of 31 markers capture genes putatively involved in the production of glucomannan, a water-soluble polysaccharide that gives salep its distinctive properties. We tested the kit on 73 taxa native to the area, demonstrating universally high locus recovery irrespective of species identity, that exceeds the total sequence length obtained with alternative kits currently available. Phylogenetic inference with concatenation and coalescent approaches was robust and showed high levels of support for most clades, including some which were previously unresolved. Resolution for hybridizing and recently radiated lineages remains difficult, but could be further improved by analysing multiple haplotypes and the non-exonic sequences captured by our kit, with the promise to shed new light on the evolution of enigmatic taxa with a complex speciation history. Offering a step-up from traditional barcoding and universal markers, the genome-wide custom loci targeted by Orchidinae-205 are a valuable new resource to study the evolution, systematics and trade of terrestrial orchids.

Because of the detrimental effects of terrestrial invasive plant species (TIPS) on native species, ecosystems, public health, and the economy, many countries have been actively looking for strategies to prevent the introduction and minimize the spread of TIPS. Fast and accurate detection of TIPS is essential to achieving these goals. Conventionally, invasive species monitoring has relied on morphological attributes. Recently, DNA-based species identification (i.e., DNA barcoding) has become more attractive. To investigate whether DNA barcoding can aid in the detection and management of TIPS, we visited multiple nature areas in Southwest Michigan and collected a small piece of leaf tissue from 91 representative terrestrial plant species, most of which are invasive. We extracted DNA from the leaf samples, amplified four genomic loci (ITS, rbcL, matK, and trnH-psbA) with PCR, and then purified and sequenced the PCR products. After careful examination of the sequencing data, we were able to identify reliable DNA barcode regions for most species and had an average PCR-and-sequencing success rate of 87.9%. We found that the species discrimination rate of a DNA barcode region is inversely related to the ease of PCR amplification and sequencing. Compared with rbcL and matK, ITS and trnH-psbA have better species discrimination rates (80.6% and 63.2%, respectively). When ITS and trnH-psbA are simultaneously used, the species discrimination rate increases to 97.1%. The high species/genus/family discrimination rates of DNA barcoding indicate that DNA barcoding can be successfully employed in TIPS identification. Further increases in the number of DNA barcode regions show little or no additional increases in the species discrimination rate, suggesting that dual-barcode approaches (e.g., ITS + trnH-psbA) might be the efficient and cost-effective method in DNA-based TIPS identification. Close inspection of nucleotide sequences at the four DNA barcode regions among related species demonstrates that DNA barcoding is especially useful in identifying TIPS that are morphologically similar to other species.

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.

We developed phyloBARCODER (https://github.com/jun-inoue/phyloBARCODER), a new web tool that can identify short DNA sequences to the species level using metabarcoding. phyloBARCODER estimates phylogenetic trees based on uploaded anonymous DNA sequences and reference sequences from databases. Without such phylogenetic contexts, alternative, similarity-based methods independently identify species names and anonymous sequences of the same group by pairwise comparisons between queries and database sequences, with the caveat that they must match exactly or very closely. By putting metabarcoding sequences into a phylogenetic context, phyloBARCODER accurately identifies (1) species or classification of query sequences and (2) anonymous sequences associated with the same species or even with populations of query sequences, with clear and accurate explanations. Version 1 of phyloBARCODER stores a database comprising all eukaryotic mitochondrial gene sequences. Moreover, by uploading their own databases, phyloBARCODER users can conduct species identification specialized for sequences obtained from a local geographic region or those of non-mitochondrial genes, e.g., ITS or rbcL.

Objectives: This study introduces MetaBIDx, a computational method designed to enhance species prediction in metagenomic environments. The method addresses the challenge of accurate species identification in complex microbiomes, which is due to the large number of generated reads and the ever-expanding number of bacterial genomes. Bacterial identification is essential for disease diagnosis and tracing outbreaks associated with microbial infections. Methods: MetaBIDx utilizes a modified Bloom filter for efficient indexing of reference genomes and incorporates a novel strategy for reducing false positives by clustering species based on their genomic coverages by identified reads. The approach was evaluated and compared with several well-established tools across various datasets. Precision, recall, and F1-score were used to quantify the accuracy of species prediction. Results: MetaBIDx demonstrated superior performance compared to other tools, especially in terms of precision and F1-score. The application of clustering based on approximate coverages significantly improved precision in species identification, effectively minimizing false positives. We further demonstrated that other methods can also benefit from our approach to removing false positives by clustering species based on approximate coverages. Conclusion: With a novel approach to reducing false positives and the effective use of a modified Bloom filter to index species, MetaBIDx represents an advancement in metagenomic analysis. The findings suggest that the proposed approach could also benefit other metagenomic tools, indicating its potential for broader application in the field. The study lays the groundwork for future improvements in computational efficiency and the expansion of microbial databases.

The Cyathocotylidae Mühling, 1898 is a family of primitive diplostomoid trematodes important for understanding the evolution of the superfamily Diplostomoidea. However, cyathocotylids remain poorly studied with the use of molecular techniques. In this study we sequenced the 5.8S + ITS2 region, 28S rRNA, and cox1 genes of two cyathocotylid species and obtained new morphological data on them. We propose Georduboisia nom. nov. instead of the preoccupied name Duboisia Szidat, 1936 (junior homonym of Duboisia Stremme, 1911). Adults of Georduboisia cf. teganuma (Ishii, 1935) and Paracoenogonimus ovatus Katsurada, 1914 were collected from fish-eating birds in the south of the European part of Russia. Georduboisia cf. teganuma was very similar to G.teganuma but differed from it in the shape of the testes. The 28S rRNA gene dataset provided the best-resolved phylogeny of the Cyathocotylidae to date. In the phylogram based on partial sequences of this gene, P. ovatus was close to members of Holostephanoides Dubois, 1983, Neogogatea Chandler & Rausch, 1947 and Gogatea Szidat, 1936. Georduboisia cf. teganuma clustered with members of Cyathocotyle Mühling, 1896 and Holostephanus Szidat, 1936. Phylogenetic analysis based on the 5.8S + ITS2 dataset showed that adults of P. ovatus examined in our study were conspecific with the metacercariae from the musculature of fish collected in Hungary and Italy. It also revealed probable misidentifications of larvae and adults of cyathocotylids whose sequences are deposited in GenBank NCBI.

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.