Method For Identification Of Species 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.

A Novel Method for Rapid Screening of Salmonidae Ingredients and Accurate Detection of Atlantic Salmon (Salmo salar) Simultaneously Using Duplex Real-Time PCR Coupled with Melting Curve Analysis

The substitution of ingredients with Salmonidae, particularly Salmo salar, has led to widespread reports of financial losses and health risks globally, emphasizing the urgent need for the development of a rapid and precise method for species identification. The aim of the present study was to develop a novel method for the rapid screening of Salmonidae ingredients and the accurate detection of S. salar simultaneously using multiplex real-time PCR coupled with melting curve analysis. Specifically, primer sets specific for S. salar and Salmonidae were cross-confirmed. Moreover, the reaction system and conditions of a real-time duplex PCR were optimized, and the proposed methodology was verified, proving that the assay has good specificity and sensitivity. Clear and distinguishable melting peaks, with expected Tm values of around 80 °C (S. salar) and 84 °C (Salmonidae), were observed for twelve products, proving the presence of S. salar. However, four products were not derived from S. salar, but they could have belonged to another species within the Salmonidae family due to the presence of only one specific melting peak at a Tm value of about 84 °C. Therefore, the novel assay in the present study allows for the fast and accurate screening of Salmonidae ingredients and the detection of S. salar simultaneously.

Comparison of Two Different DNA Isolation Methods (Quick & Dirty vs. Commercial Kit) in Small Insects

The COI gene region is the most commonly employed method for species identification in insects, as in all animals. The importance of DNA isolation methods is increasing, and they are of great importance for identifying insects. Unlike other large animals, DNA isolation from insects, especially from agricultural pests with very small structures, is possible with expensive commercial kits. In this study, an expensive commercial kit and a fast and economical method were compared in terms of time, cost, DNA quantity and quality. It was found that the success of commercial kits in terms of DNA quality and quantity is unquestionable. However, it was determined that the modified ‘quick and dirty’ method can be successfully used to extract DNA from insects and insect parts of all sizes, especially in studies that do not require long-term storage of DNA. Average isolation time for Q&D was 45 minutes, compared to 4 hours or more for commercial kits. Finally, cost is the most important factor in today’s global economy. The unit cost of DNA isolation using the Q&D method is approximately 88% lower.

Assembly-free reads accurate identification (AFRAID) approach outperforms other methods of DNA barcoding in the walnut family (Juglandaceae)

DNA barcoding has been extensively used for species identification. However, species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods. In this study, we use plant species in Juglandaceae to evaluate an assembly-free accurate reads identification (AFRAID) method of species identification, a novel approach for precise species identification in plants. Specifically, we determined (1) the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae, (2) the minimum size of chloroplast dataset for species discrimination, and (3) minimum amount of next generation sequencing (NGS) data required for species identification. We found that species identification rates were highest when whole chloroplast genomes were used, followed by taxon-specific DNA barcodes, and then universal DNA barcodes. Species identification of 100% was achieved when chloroplast genome sequence coverage reached 20% and the original sequencing data reached 500,000 reads. AFRAID accurately identified species for all samples tested after 500,000 clean reads, with far less computing time than common approaches. These results provide a new approach to accurately identify species, overcoming limitations of traditional DNA barcodes. Our method, which uses next generation sequencing to generate partial chloroplast genomes, reveals that DNA barcode regions are not necessarily fixed, accelerating the process of species identification.

Robust mosquito species identification from diverse body and wing images using deep learning

Mosquito-borne diseases are a major global health threat. Traditional morphological or molecular methods for identifying mosquito species often require specialized expertise or expensive laboratory equipment. The use of convolutional neural networks (CNNs) to identify mosquito species based on images may offer a promising alternative, but their practical implementation often remains limited. This study explores the applicability of CNNs in classifying mosquito species. It compares the efficacy of body and wing depictions across three image collection methods: a smartphone, macro-lens attached to a smartphone and a professional stereomicroscope. The study included 796 specimens of four morphologically similar Aedes species, Aedes aegypti, Ae. albopictus, Ae. koreicus and Ae. japonicus japonicus. The findings of this study indicate that CNN models demonstrate superior performance in wing-based classification 87.6% (95% CI: 84.2–91.0) compared to body-based classification 78.9% (95% CI: 77.7–80.0). Nevertheless, there are notable limitations of CNNs as they perform reliably across multiple devices only when trained specifically on those devices, resulting in an average decline of mean accuracy by 14%, even with extensive image augmentation. Additionally, we also estimate the required training data volume for effective classification, noting a reduced requirement for wing-based classification compared to body-based methods. Our study underscores the viability of both body and wing classification methods for mosquito species identification while emphasizing the need to address practical constraints in developing accessible classification systems.Graphical abstract

Pest categorisation of non-EU Scolytinae on non-coniferous hosts.

The EFSA Panel on Plant Health performed a group pest categorisation for the EU territory of non-EU Scolytinae (Coleoptera: Curculionidae) on non-coniferous hosts, which total 6495 known species. Most species attack apparently healthy, weakened or dead trees, either feeding on the phloem ('bark beetles' subgroup) or on fungi inoculated into the sapwood ('ambrosia beetles' subgroup). Smaller subgroups feed and reproduce in seeds and fruits, or in herbaceous plants. Some species are polygynous, the males initiate a gallery or a chamber on or in a new host and attract females. Others are monogamous, and the females initiate the new galleries. Many species respond to primary volatile attractants emitted by the hosts, and some produce aggregation pheromones that attract conspecifics of both sexes. The species attacking living hosts are often associated with fungi that contribute to weakening the host defences and provide nutrients to the insects. Some are inbreeding; the males in the offspring mate with their sisters and rarely leave their natal tree. The larvae of all species develop and pupate within their hosts. Based on catalogues and other published data, a database was constructed providing information on hosts, feeding and reproductive habits, geographic distribution and the Köppen-Geiger climate types in countries where species occur. The Scolytinae were screened to exclude species in the following categories: (i) 708 species attacking conifers; (ii) 127 species present in at least four EU Member States and (iii) 440 species occurring in areas with climatic conditions not occurring in the EU. Among the remaining 5220 species, 88 species known for their mobility, occupying at least two landmasses separated by geographical barriers and some of which had impact levels documented in literature, were extracted. They were grouped into four subcategories: (i) 12 species with high impact on plant health; (ii) 16 species with low or doubtful impact; (iii) 48 species with no impact; (iv) 12 species with no impact and which had never been recorded as 'introduced' in the consulted catalogues but occurring on at least two landmasses. All 88 species could enter the EU with wood or wood products, or with plants for planting, and could establish because host plants are available, and climate is suitable in parts of the EU. Control measures to inhibit introduction are available. There is considerable uncertainty regarding the potential impact of many species. Methods for the reliable identification of many species are lacking. For some species of non-EU Scolytinae on non-coniferous hosts, all criteria assessed by EFSA for consideration as potential quarantine pest are met. Nevertheless, the Panel was not able to develop a method to discriminate confidently between species that clearly meet the criteria for potential quarantine pest status and those that do not.

DNA Barcoding of the Endemic Freshwater Catfish Encheloclarias tapeinopterus (Bleeker, 1853) (Siluriformes:Clariidae) from Bangka Island, Indonesia

Abstract Encheloclarias tapeinopterus, an endemic catfish exclusively found in Bangka Island, Indonesia. The population of this species has been observed to be decreasing at an alarming rate, which has resulted in the IUCN Red List classifying it as a species that is vulnerable (VU). During our expedition on the 25th and 26th of January 2023, a total of Two specimens of E. tapeinopterus were collected from Kelumpik River, Pinang Sebatang Village, Central Bangka Regency, Bangka Island, Indonesia. In this groundbreaking study, we proudly present the pioneering documentation of E. tapeinopterus DNA barcoding, focusing on the Cytochrome C oxidase Subunit I (COI) gene. The recorded DNA Barcode of E. tapeinopterus has been officially registered in the Genbank under the accession code OQ281707. By employing DNA barcoding, we offer an advanced and expedient method for precise species identification and discovery, revolutionizing the field of taxonomy. Phylogenetic analysis revealed that E. tapeinopterus forms a distinct clade alongside the Clarias and Heterobranchus genus, shedding light on its evolutionary relationships and genetic affinities.

Development of a diagnostic variable number tandem repeat marker and dual TaqMan genotyping assay to distinguish Lophophora species.

The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii, which has traditionally been used as a natural analgesic; however, its use is now under strict regulation worldwide as it contains mescaline, a unique psychotropic agent. Recently, non-medical and illegal distribution and abuse of L. williamsii have increased worldwide; thus, effective species identification methods are urgently needed. Here, we identified a new variable number tandem repeat (VNTR) marker in the trnL intron region to identify and characterize species in forensic analyses. The VNTR marker has a unique structure of tandem repeats, each with 13 nucleotides; one repeat unit was found in L. williamsii and two in L. diffusa. Phylogenetic and length polymorphism analyses confirmed that this novel VNTR marker could distinguish between Lophophora species. Furthermore, our newly developed TaqMan genotyping assay utilizes two probes; the color and position of dots on the discrimination plot differ according to the tandem repeat count within the VNTR marker. The limits of detection of the assay were 0.000063ng (LW-VNTR probe-1) and 0.000066ng (LW-VNTR probe-2), indicating high sensitivity. Moreover, when crime scene samples of 16 presumed L. williamsii species were analyzed, the results coincided with those of gas chromatography-mass spectrometry, confirming the applicability of our marker for Lophophora species identification. Thus, the tandem repeats within the trnL intron region can be exploited as a VNTR marker to identify L. williamsii and L. diffusa. Our dual TaqMan genotyping assay based on a novel marker demonstrates potential for forensic applications.

Multi-marker DNA metabarcoding for precise species identification in ichthyoplankton samples

Ichthyoplankton monitoring is crucial for stock assessments, offering insights into spawning grounds, stock size, seasons, recruitment, and changes in regional ichthyofauna. This study evaluates the efficiency of multi-marker DNA metabarcoding using mitochondrial cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA gene markers, in comparison to morphology-based methods for fish species identification in ichthyoplankton samples. Two transects with four coastal distance categories were sampled along the southern coast of Portugal, being each sample divided for molecular and morphological analyses. A total of 76 fish species were identified by both approaches, with DNA metabarcoding overperforming morphology—75 versus 11 species-level identifications. Linking species-level DNA identifications with higher taxonomic morphological identifications resolved several uncertainties associated with traditional methods. Multi-marker DNA metabarcoding improved fish species detection by 20–36% compared to using a single marker/amplicon, and identified 38 species in common, reinforcing the validity of our results. PERMANOVA analysis revealed significant differences in species communities based on the primer set employed, transect location, and distance from the coast. Our findings underscore the potential of DNA metabarcoding to assess ichthyoplankton diversity and suggest that its integration into routine surveys could enhance the accuracy and comprehensiveness of fish stock assessments.

Species identification of Panax ginseng throughout the entire industrial chain: From the ginseng field to highly processed products

Panax ginseng has been highly treasured in Asian countries due to its long history, more types of pharmaceutically active ginsenosides, and prominent pharmacological effects. However, the potential adulteration of P. ginseng with P. quinquefolius or P. notoginseng poses serious health risks to consumers and disrupts market stability within the ginseng industry. To address this challenge, a simple real-time PCR assay for rapid screening of field P. ginseng and a reliable multiplex PCR method for species identification of highly processed ginseng products were respectively established, based on the InDel markers derived from nuclear and mitochondrial intron sequences. The real-time PCR assay, coupled with a simple NaOH-Tris DNA extraction method, facilitates high-throughput screening of field ginseng samples based on the Tm values of PCR products with a single pair of primers. The multiplex PCR assay, targeting the mitochondrial nad1 and nad5 InDel markers, could detect exogenous adulteration of P. quinquefolius or P. notoginseng at a remarkable detection limit of 0.001 ng and successfully identified the original species in various forms of highly processed ginseng products. The results indicate that mitochondrial InDel markers resist DNA degradation better than nuclear ones, making them more suitable for species identification in highly processed ginseng products that contain trace amounts of degraded DNA. The present study provides robust InDel marker-based methods for species identification across the entire ginseng industrial chain, from the ginseng field to final processed products, thereby serving as reliable tools for ensuring the authenticity of P. ginseng products.

Fish eggs in the diet of White Sea cod Gadus morhua marisalbi (Gadiformes: Gadidae): species identification using DNA-based methods

Trophic relationships reflect the peculiarities of the functioning of communities of aquatic organisms and analysis of their structure and dynamics can be used to improve fisheries management and assess the state of marine ecosystems. Populations at the margins of the species' range have particular adaptations, including feeding habits, to extreme conditions, which makes them a convenient model object for studying the possible consequences of ecosystem changes under the influence of global factors. The cod Gadus morhua marisalbi Derjugin, 1920 permanently inhabits the coastal waters of the subarctic White Sea, located at the northeastern margin of the geographic range of the Atlantic cod G. morhua Linnaeus, 1758. This coastal cod population strongly depends on the local environmental conditions and available food resources during the summer feeding period. In addition to the main food organisms (fish, polychaetes, crustaceans), the eggs of fish spawning in shallow coastal areas in summer are also important in the diet of cod at this time. According to published data, fish eggs found in significant quantities and most often in the stomachs of cod belonged to threespine stickleback and lumpfish, but this has not been confirmed by any experimental research. Besides, the possible presence of herring eggs in the diet of cod remains unproven due to the difficulties of its visual identification. In this work, we used molecular genetic methods for species identification of fish eggs found in cod stomachs. At the first stage, the species identification of DNA isolated from eggs was done using two sets of species-specific primers: the first which we designed for the mitochondrial COI gene of stickleback, cod, and herring, and the second, published for a microsatellite locus of herring. At the second stage, the species identity of DNA extracted from fish eggs was checked using DNA barcoding. The results showed that cod stomachs contained herring and stickleback eggs, and in a number of cases we found the co-occurrence of eggs of these species. Of the 29 DNA preparations studied, fourteen were successfully amplified with microsatellite primers for herring, and thirteen amplified with primers for stickleback. Interannual differences in the occurrence of herring eggs in samples were insignificant, while the frequency of occurrence of stickleback eggs in 2018 was higher than in 2017. Our analysis did not reveal significant intra-annual differences between the observed and theoretically expected number of cases of simultaneous occurrences of herring and stickleback eggs. The high frequency of occurrence of herring and stickleback eggs in cod stomachs revealed in this study indicates the importance of these components in the diet of cod and provides new knowledge on the trophic role of cod as a key species in the White Sea ecosystem. Analysis of available data on the spatial and temporal diet patterns of coastal White Sea cod shows that fish eggs can significantly contribute to the total amount of food consumed by this species during the summer.

Enterobacter adelaidei sp. nov. Isolation of an extensively drug resistant strain from hospital wastewater in Australia and the global distribution of the species

BackgroundEnterobacter species are included among the normal human gut microflora and persist in a diverse range of other environmental niches. They have become important opportunistic nosocomial pathogens known to harbour plasmid-mediated multi-class antimicrobial resistance (AMR) determinants. Global AMR surveillance of Enterobacterales isolates shows the genus is second to Klebsiella in terms of frequency of carbapenem resistance. Enterobacter taxonomy is confusing and standard species identification methods are largely inaccurate or insufficient. There are currently 27 named species and a total of 46 taxa in the genus distinguishable via average nucleotide identity (ANI) calculation between pairs of genomic sequences. Here we describe an Enterobacter strain, ECC3473, isolated from the wastewater of an Australian hospital whose species could not be determined by standard methods nor by ribosomal RNA gene multi-locus typing. AimTo characterise ECC3473 in terms of phenotypic and genotypic antimicrobial resistance, biochemical characteristics and taxonomy as well as to determine the global distribution of the novel species to which it belongs. MethodsStandard broth dilution and disk diffusion were used to determine phenotypic AMR. The strain’s complete genome, including plasmids, was obtained following long- and short read sequencing and a novel long/short read hybrid assembly and polishing, and the genomic basis of AMR was determined. Phylogenomic analysis and quantitative measures of relatedness (ANI, digital DNA-DNA hybridisation, and difference in G+C content) were used to study the taxonomic relationship between ECC3473 and Enterobacter type-strains. NCBI and PubMLST databases and the literature were searched for additional members of the novel species to determine its global distribution. ResultsECC3473 is one of 21 strains isolated globally belonging to a novel Enterobacter species for which the name, Enterobacter adelaidei sp. nov. is proposed. The novel species was found to be resilient in its capacity to persist in contaminated water and adaptable in its ability to accumulate multiple transmissible AMR determinants. ConclusionE. adelaidei sp. nov. may become increasingly important to the dissemination of AMR.

Morphometric Characters and Meristic Counts of Mastacembelus armatus (Lacebede, 1800) from Maruthur Anicut, Tirunelveli District, India

The present study highlights about morphometric measurement and meristic count of Mastacembelus armatus (Lacebede, 1800) collected from the Maruthur wetland ecosystem have been investigated with 23 specimens to observe the relationship between morphometric characters. Identification of species is an initial step in any research work and plays a vital role in the behavioural study. Morphometric character analysis and meristic counts are considered as simplest and authentic methods for fish species identification which is termed as morphological systematics. Fourteen different morphometric characters in percentage of total fish length (9 characters), head length (5 characters), and five different meristic counts (branched and unbranched rays) have been studied. The obtained result reveals that morphometric characters vary depending on the growth and size of the fish. However, the meristic counts were comparatively similar. All the characters show a positive correlation both in the percentage of total length and head length. The different morphometric and meristic counts of M. armatus have been analyzed for the population dynamics which includes taxonomy study and proper identification of fishes. The fin formula was calculated for some hard rays and soft rays. Therefore, this study supplement additional data for the identification of the fish species.

Comparative analysis of complete chloroplast genomes of Synotis species (Asteraceae, Senecioneae) for identification and phylogenetic analysis

BackgroundThe Synotis (C. B. Clarke) C. Jeffrey & Y. L. Chen is an ecologically important genus of the tribe Senecioneae, family Asteraceae. Because most species of the genus bear similar morphology, traditional morphological identification methods are very difficult to discriminate them. Therefore, it is essential to develop a reliable and effective identification method for Synotis species. In this study, the complete chloroplast (cp.) genomes of four Synotis species, S. cavaleriei (H.Lév.) C. Jeffrey & Y.L. Chen, S. duclouxii (Dunn) C. Jeffrey & Y.L. Chen, S. nagensium (C.B. Clarke) C. Jeffrey & Y.L. Chen and S. erythropappa (Bureau & Franch.) C. Jeffrey & Y. L. Chen had been sequenced using next-generation sequencing technology and reported here.ResultsThese four cp. genomes exhibited a typical quadripartite structure and contained the large single-copy regions (LSC, 83,288 to 83,399 bp), the small single-copy regions (SSC, 18,262 to 18,287 bp), and the inverted repeat regions (IR, 24,837 to 24,842 bp). Each of the four cp. genomes encoded 134 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes (ycf1 and rps19). The highly variable regions (trnC-GCA-petN, ccsA-psaC, trnE-UUC-rpoB, ycf1, ccsA and petN) may be used as potential molecular barcodes. The complete cp. genomes sequence of Synotis could be used as the potentially effective super-barcode to accurately identify Synotis species. Phylogenetic analysis demonstrated that the four Synotis species were clustered into a monophyletic group, and they were closed to the Senecio, Crassocephalum and Dendrosenecio in tribe Senecioneae.ConclusionsThis study will be useful for further species identification, evolution, genetic diversity and phylogenetic studies within this genus Synotis and the tribe Senecioneae.

Genomic oropharyngeal Neisseria surveillance detects MALDI-TOF MS species misidentifications and reveals a novel Neisseria cinerea clade.

Introduction. Commensal Neisseria spp. are highly prevalent in the oropharynx as part of the healthy microbiome. N. meningitidis can colonise the oropharynx too from where it can cause invasive meningococcal disease. To identify N. meningitidis, clinical microbiology laboratories often rely on Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS).Hypothesis/Gap statement. N. meningitidis may be misidentified by MALDI-TOF MS.Aim. To conduct genomic surveillance of oropharyngeal Neisseria spp. in order to: (i) verify MALDI-TOF MS species identification, and (ii) characterize commensal Neisseria spp. genomes.Methodology. We analysed whole genome sequence (WGS) data from 119 Neisseria spp. isolates from a surveillance programme for oropharyngeal Neisseria spp. in Belgium. Different species identification methods were compared: (i) MALDI-TOF MS, (ii) Ribosomal Multilocus Sequence Typing (rMLST) and (iii) rplF gene species identification. WGS data were used to further characterize Neisseria species found with supplementary analyses of Neisseria cinerea genomes.Results. Based on genomic species identification, isolates from the oropharyngeal Neisseria surveilence study were composed of the following species: N. meningitidis (n=23), N. subflava (n=61), N. mucosa (n=15), N. oralis (n=8), N. cinerea (n=5), N. elongata (n=3), N. lactamica (n=2), N. bacilliformis (n=1) and N. polysaccharea (n=1). Of these 119 isolates, four isolates identified as N. meningitidis (n=3) and N. subflava (n=1) by MALDI-TOF MS, were determined to be N. polysaccharea (n=1), N. cinerea (n=2) and N. mucosa (n=1) by rMLST. Phylogenetic analyses revealed that N. cinerea isolates from the general population (n=3, cluster one) were distinct from those obtained from men who have sex with men (MSM, n=2, cluster two). The latter contained genomes misidentified as N. meningitidis using MALDI-TOF MS. These two N. cinerea clusters persisted after the inclusion of published N. cinerea WGS (n=42). Both N. cinerea clusters were further defined through pangenome and Average Nucleotide Identity (ANI) analyses.Conclusion. This study provides insights into the importance of genomic genus-wide Neisseria surveillance studies to improve the characterization and identification of the Neisseria genus.

Biological Characteristics, Artificial Domestication Conditions Optimization, and Bioactive Components of Beauveria caledonica.

In this study, one strain of Beauveria caledonica was isolated from wild fruiting bodies collected from Guizhou Province, China, and its species identification, biological characteristics, domestication, and cultivation methods were studied along with polysaccharide and adenosine content analysis. The mycelia were identified by ITS sequencing, and the fruiting bodies of B. caledonica were domestically cultivated for the first time using wheat and rice as basic cultivation media. The carbon sources, nitrogen sources, cultivation temperatures, and pH for mycelial growth were optimized through single-factor experiments and response surface methodology (RSM) experiments. The polysaccharide content was detected by the phenol-sulfuric acid method, and the adenosine content was measured by high-performance liquid chromatography (HPLC). The results confirmed that the identified mycelia were B. caledonica. The optimum medium for solid culture was 25.8 g/L glycerol, 10.9 g/L yeast extract, 1 g/L MgSO4·7H2O, 1 g/L KH2PO4, 10 mg/L vitamin B1, and 20 g/L agar; the optimum pH was 6.5, and the optimum culture temperature was 25 °C. The optimal liquid culture medium was 26.2 g/L glycerol, 11.1 g/L yeast extract, 1 g/L MgSO4·7H2O, 1 g/L KH2PO4, and 10 mg/L vitamin B1; the mycelia grew well at pH 6.6 and 25 °C. The average biological efficiencies of fruiting bodies on wheat and rice as culture media were 1.880% and 2.115%, respectively; the polysaccharide contents of fruiting bodies on the two media were 6.635% and 9.264%, respectively, while the adenosine contents were 0.145% and 0.150%, respectively. This study provides a valuable reference for further artificial cultivation and utilization of B. caledonica by investigating its biological characteristics, cultivation conditions for artificial domestication, and polysaccharide and adenosine contents in cultivated fruiting bodies.

Biogeography of larval trematodes in the freshwater snail, Semisulcospira libertina: a comparison of the morphological and molecular approaches

While biogeographic patterns of free-living organisms are well documented, the biogeography of parasitic fauna remains largely unclear. Due to morphological similarities, parasites are often difficult to identify without the aid of molecular genetics, further complicating the interpretation of their biogeographic patterns. We investigated trematode parasites infecting the East Asian freshwater snail Semisulcospira libertina to understand their biogeography and to evaluate how molecular approaches influence the interpretation of biogeographic patterns of the trematode fauna. We identified 46 genetically delimited species from 19 morphologically distinguishable trematodes infecting S. libertina and found that their species richness was negatively correlated to latitude. We also found that potential definitive host (fishes) richness and host body size were positively correlated with trematode species richness, suggesting that host attributes are essential factors shaping the biogeographic pattern in trematodes. These trends were observed irrespective of species identification methods, demonstrating that classical morphological identification can also effectively identify the latitudinal gradient pattern in trematodes. We further detected the distance decay of similarity in trematode communities, although this trend was only detectable in the biogeographic dataset based on molecular identification. Our study showed that morphological identification sufficiently reflects the latitudinal richness gradient while molecular identification is essential to estimate accurate local species richness and increase the resolution of the large-scale pattern of population similarities in the trematode communities.

Molecular characterization of Armigeres Subalbatus from Hyderabad region of Telangana state, India.

The mosquito Armigeres subalbatus (Coquillett, 1898) is a significant vector for Japanese encephalitis infection, and breeds in high organic polluted water. Understanding mosquito diversity and there abundance in relation to mosquito-borne diseases is an important component for public health managers. Though the conventional methods for systematic position of mosquito species by using morphological characteristics is a classical method, but it requires perfect expertise and well preserved specimen. Conversely, the molecular analysis of mitochondrial cytochrome C oxidase subunit I (COI) serves as a gene-centric DNA barcoding approach and offers a promising alternative method for mosquito species identification. The study at hand delves into the morphological characteristics of Armigeres subalbatus were compared with COI- gene to ensure a more dependable verification for identification of mosquito species found in Hyderabad region of Telangana. The 489 base pair amplicons were acquired and deposited into the NCBI Gene Bank nucleotide database under the accession number MG686500. The maximum likelihood tree infers that, the Hyderabad species was diverged from USA and Japan species but had ancestral relationship with Tamil Nadu, Karnataka, Maharastra, Kerala and Goa species. Mitochondrial gene (COI) based DNA barcoding is the most reliable and potential alternative technique to identify the mosquito species.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang etal. 1993.

Identification of Nontuberculous Mycobacterium Species by Polymerase Chain Reaction - Restriction Enzyme Analysis (PCR-REA) of rpoB gene in Clinical Isolates.

Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.