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Simple Summary: The Danube Clouded Yellow (Colias myrmidone) is a species that is under special conservation concern in Europe. The reasons for its quick and dramatic decline and local extirpations are not fully understood; genetic data are vital to understanding these reasons. We studied the last two neighboring populations from Central Europe—one that was recently extirpated and another that still exists—using a mitochondrial barcoding marker and a nuclear gene. We also tested samples for the endosymbiotic bacterium Wolbachia, which is known to induce reproductive manipulations in their hosts, affecting mtDNA diversity among populations. The results of our research suggest that a relatively high genetic polymorphism has decreased in recent years. Restricted gene flow that is related to habitat fragmentation and isolation has led to the quick differentiation of populations; however, these populations probably used to be genetically uniform, at least at the regional scale. DNA barcode-based analysis revealed the presence of two haplogroups, but, interestingly, only individuals from one of them were infected by Wolbachia. This phenomenon turned out to be female-biased, which has a complex impact on the host. Finally, a comparison of our data with other available data indicated significant barcode sharing between C. myrmidone and its sibling C. caucasica, which allows us to question the distinctiveness of both vulnerable taxa. The Danube Clouded Yellow (Colias myrmidone) has experienced one of the most dramatic declines among European butterflies. To estimate genetic diversity in the last population in Poland that has survived in the Knyszyn Forest (KF), we analyzed mitochondrial (COI) and nuclear (EF-1α) polymorphisms in individuals sampled in 2014 and 2022. The results were compared with genetic data obtained in 2014 from a recently extirpated nearby population (Czerwony Bór, CB). Because mtDNA polymorphisms in insects can be modulated by endosymbionts, the samples were screened for Wolbachia. The polymorphism of EF-1α indicated that diversity was gradually decreasing. The KF experienced rapid demographic processes, manifested by a significant change in allele frequency. The small differentiation in nuclear markers between the KF and CB in 2014 suggests that the regional population used to be genetically uniform. Four COI haplotypes that were identified in this study probably belong to two different haplogroups. Wolbachia was detected only in individuals with one specific haplotype, and the prevalence was female-biased, suggesting the induction of two reproductive manipulations. The most common COI haplotype found in Poland was the same as that reported from other parts of Europe, not only for C. myrmidone but also C. caucasica. These results allow us to question the distinctiveness of each taxa. [ABSTRACT FROM AUTHOR]

Simple Summary: Safflower is a valuable crop used in various industries, but it suffers from pest attacks. Recently, Condica capensis, a pest previously unreported in China, was found in Yunnan Province. Our study examines its physical and biological traits, life cycle, and natural enemies. By identifying and understanding this pest, we aim to help farmers and researchers effectively manage and control its spread, ensuring healthy safflower cultivation. Condica capensis (Lepidoptera: Noctuidae), a newly identified pest in Yunnan Province, China, poses a threat to safflower crops. Discovered in Nanhua County in November 2023, the pest damages safflower at multiple life stages, especially during its larval stage, when it feeds on leaves, tender stems, and flower filaments, sometimes causing the entire plant to die. Morphological and molecular analyses, including mitochondrial cytochrome C oxidase I (COI) gene sequencing, confirmed its identity as C. capensis, a new species record for Yunnan. The study also documented the pest's life cycle, reproductive behavior, and natural enemies, highlighting the potential for biological control using parasitic wasps such as Cotesia sp. This research emphasizes the need for accurate pest identification and monitoring to develop effective, sustainable pest management strategies. As safflower cultivation grows in Yunnan, managing C. capensis is critical to safeguarding local agriculture and preventing broader agricultural threats. [ABSTRACT FROM AUTHOR]

Traditionally, classifying species has required taxonomic experts to carefully examine unique physical characteristics, a time-intensive and complex process. Machine learning offers a promising alternative by utilizing computational power to detect subtle distinctions more quickly and accurately. This technology can classify both known (described) and unknown (undescribed) species, assigning known samples to specific species and grouping unknown ones at the genus level—an improvement over the common practice of labeling unknown species as outliers. In this paper, we propose a novel ensemble approach that integrates neural networks with support vector machines (SVM). Each animal is represented by an image and its DNA barcode. Our research investigates the transformation of one-dimensional vector data into two-dimensional three-channel matrices using discrete wavelet transform (DWT), enabling the application of convolutional neural networks (CNNs) that have been pre-trained on large image datasets. Our method significantly outperforms existing approaches, as demonstrated on several datasets containing animal images and DNA barcodes. By enabling the classification of both described and undescribed species, this research represents a major step forward in global biodiversity monitoring. [ABSTRACT FROM AUTHOR]

Trigonella foenum-graecum (L.) is one of the potent medicinal plants and is a well-known spice for its specific aroma. It is a native plant of Kasur District of Punjab Pakistan commonly known as Kasuri methi. Kasur district is a semi-arid subtropical area, famous for its production worldwide. The exact plant identification up to the species level is hard for plant taxonomists and herbalists, due to its resemblance with its close relatives. DNA barcoding is the most modern approach for plant identification based upon a highly conserved chloroplast genome. Rbcla marker is considered one of the most conserved regions used for highly complex and confusing species-level identification. The current study is the first effort for molecular identification of Trigonella foenum-graecum (L.) based upon DNA barcoding in South Asia. Chloroplast DNA was isolated by applying the modified CTAB method. Specific primers for rbcLa were used for polymerase chain reaction (PCR) amplification. Purified PCR products were then subjected to DNA sequence for rbcla DNA barcode. After DNA sequencing, the chromatogram file was analyzed by using Chromas. Then align sequences by multiple sequence alignment by using clustalW in Geneious. Afterward, BLAST the sequence from the NCBI database and align this query sequence. The rbcLa barcode showed 99.80% precision for species identification. The cataloging derivative from the DNA barcode tree was then compared with archaic morphology-based taxonomy. This will help identification of Kasuri methi from its closely related species for better business opportunities and safer usage by the public as spice and medicine. [ABSTRACT FROM AUTHOR]

Background: The Tephritidae family, commonly referred to as true fruit flies, comprises of a substantial group within order Diptera. Numerous species within this family are major agricultural pests, with a tendency to infest a wide array of fruits and vegetables in tropical and sub- tropical regions, leading to considerable damage and consequent reductions in the market value of the crops. Methods and results: The current study was aimed to propose a promising solution to the menace posed by fruit flies by offering rapid, accurate and reliable species identification by using character-based DNA barcode methodology. The Tephritid specimens were collected from Cucurbitaceous plants of southern parts of West Bengal, India, and a total of eight species from Tephritidae family were obtained belonging to three genera, namely Bactrocera (Macquart, 1835), Dacus (Fabricius, 1805) and Zeugodacus (Hendel, 1927). Their morphological features were meticulously studied based on available literature, along with genetic analysis based on mitochondrial COI and ND1 gene sequences. A total of 30 uniquely variable sites at nucleotide position 42,48,51,60,66,72, 105,111,144,198,207,243, 273,297,307,318,345,357, 375,378,381,387,399,400, 402,436,444,450,453 and 460 in COI gene were discerned among Tephritid species in the present study. Conclusions: The character-based DNA barcode holds the potential to differentiate closely related species of fruit flies and morphologically look-a-like ones. The novel method will be very significant in terms of rapid, precise and reliable species identification and might be extremely essential for early detection during pest outbreaks by facilitating timely intervention strategies to mitigate crop damage. [ABSTRACT FROM AUTHOR]

This study aims to explore the diversity of land snails in a forested area of the second largest Brazilian city (Rio de Janeiro), through the study of basic morphology (ie shell, operculum, jaw and radula) and the barcode of COI sequences. The conservation unit 'Parque Natural Municipal da Cidade' (PNM da Cidade) is a fragment of the Atlantic Forest biome. During 12 fieldwork visits, 17 species of terrestrial gastropods were found in PNM da Cidade, of which six are synanthropic, exotic/cryptogenic, and occurred in single or few sampling stations, whereas the 11 native species varied in their range. PNM da Cidade houses a large population of the native species Thaumastus cf. taunaisii (Férussac, 1822), always found close to water sources, but it demands conservation concern. The sampling of live specimens allowed the study of operculum, jaw or radula from five selected species, and the generation of 20 COI sequences from 10 species, elucidating taxonomic aspects. The gastropod fauna at PNM da Cidade is indicative of moderately preserved habitats, despite some anthropic pressure. Future geography-based studies of land snails could add DNA sequences in order to enable the discovery or elucidation of cryptic complexes of species. [ABSTRACT FROM AUTHOR]

Low coverage 'genome‐skims' are often used to assemble organelle genomes and ribosomal gene sequences for cost‐effective phylogenetic and barcoding studies. Natural history collections hold invaluable biological information, yet poor preservation resulting in degraded DNA often hinders polymerase chain reaction‐based analyses. However, it is possible to generate libraries and sequence the short fragments typical of degraded DNA to generate genome‐skims from museum collections. Here we introduce a snakemake toolkit comprised of three pipelines skim2mito, skim2rrna and gene2phylo, designed to unlock the genomic potential of historical museum specimens using genome skimming. Specifically, skim2mito and skim2rrna perform the batch assembly, annotation and phylogenetic analysis of mitochondrial genomes and nuclear ribosomal genes, respectively, from low‐coverage genome skims. The third pipeline gene2phylo takes a set of gene alignments and performs phylogenetic analysis of individual genes, partitioned analysis of concatenated alignments and a phylogenetic analysis based on gene trees. We benchmark our pipelines with simulated data, followed by testing with a novel genome skimming dataset from both recent and historical solariellid gastropod samples. We show that the toolkit can recover mitochondrial and ribosomal genes from poorly preserved museum specimens of the gastropod family Solariellidae, and the phylogenetic analysis is consistent with our current understanding of taxonomic relationships. The generation of bioinformatic pipelines that facilitate processing large quantities of sequence data from the vast repository of specimens held in natural history museum collections will greatly aid species discovery and exploration of biodiversity over time, ultimately aiding conservation efforts in the face of a changing planet. [ABSTRACT FROM AUTHOR]

Herein, we describe three new species of the spider genus Naphrys Edwards, 2003 from Mexico: Naphrys echerisp. nov., Naphrys tecoxquinsp. nov., and Naphrys tuucasp. nov. An integrative taxonomic approach was applied, utilizing data from morphology, ultra-morphology, the mitochondrial gene COI, and distribution records. Four molecular methods for species delimitation were implemented under the corrected p-distance Neighbor-Joining (NJ) criteria: (1) Assemble Species by Automatic Partitioning (ASAP); (2) general mixed Yule coalescent (GMYC); (3) Bayesian Poisson tree process (bPTP); and (4) multi-rate Poisson tree process (mPTP). Both morphological and molecular data supported the delimitation and recognition of the three new species. The average interspecific genetic distance (p-distance) within the genus Naphrys is 14%, while the intraspecific genetic distances (p-distance) is <2% for most species. We demonstrate that the natural distribution of Naphrys is not restricted to the Nearctic region. Furthermore, the reported localities herein represent the first with precise locations in the country for Naphrys acerba. In addition, a taxonomic identification key is provided for the species in the genus. [ABSTRACT FROM AUTHOR]

Abstract Background Culex tritaeniorhynchus has long been considered the primary vector of Japanese encephalitis virus (JEV), but until recently, it was considered exotic to Australia. When the species was detected in the country’s Northern Territory (NT) for the first time, the Western Australia (WA) Department of Health was cognisant of the risk it posed to the State because of the shared border and continuous mosquito habitat adjoining the two jurisdictions. The aim of this study was to undertake intensive mosquito surveillance in the Kimberley region to ascertain whether Cx. tritaeniorhynchus was present in WA, define the extent of its distribution and undertake phylogenetic analysis of select specimens to support hypothesized routes of entry into the state. Methods Carbon dioxide (CO2)-baited encephalitis virus surveillance (EVS) mosquito traps were deployed at various sites throughout the Kimberley region by surveillance officers within the Medical Entomology unit of the Western Australia (WA) Department of Health. Mosquitoes were then morphologically identified, and a subset of four specimens were confirmed as Cx. tritaeniorhynchus by molecular identification using Cytochrome Oxidase I (COI) DNA data and phylogenetic analysis. Results From 31 March 2021 to 30 May 2024, a total of 211 female Cx. tritaeniorhynchus specimens were collected from 21 unique trap sites in the Kimberley’s Shire of Wyndham-East Kimberley (SWEK). Four COI DNA barcode regions were amplified and successfully sequenced for analysis. These sequences fell within a clade recognised as Cx. tritaeniorhynchus and specifically all sequences were in a clade with other specimens from the NT and Timor-Leste. Conclusions This study represents the first detection of Cx. tritaeniorhynchus in WA. Given the widespread nature of trap sites that yielded the species and consecutive seasons over which it was observed, the authors surmise that Cx. tritaeniorhynchus is now established within the northeast Kimberley region. The findings are significant given the detection of the species coincides with the first significant outbreak of JEV activity on mainland Australia involving an estimated 45 human cases of Japanese encephalitis, 80 impacted commercial piggeries and widespread feral pig activity. Although the role that Cx. tritaeniorhynchus may play in JEV transmission into the future is not yet understood, it presents a potential risk to public health in the region. Graphical abstract

Dry rainforest communities are globally threatened by anthropogenic pressures and climatic change but are less well researched and more poorly conserved than mesic rainforests. In response to the increasing loss of biodiversity, the Australian Government joined other international signatory parties to adopt the Kunming-Montreal Global Biodiversity Framework (GBF). The GBF emphasises the maintenance of connectivity and genetic diversity of whole ecosystems via landscape-scale conservation initiatives. Rainforest plant diversity, distinctiveness, and the current level of conservation of seasonal rainforest regional ecosystems of the Central Queensland Coast region in Australia were evaluated. Our three-marker DNA barcode dated phylogeny of rainforest plant taxa together with community species lists were used to calculate phylogenetic diversity (PD) estimates and species composition. Levels of rainforest ecosystem protection were assessed using Queensland government data. This study found selection pressures for moisture and geology significantly influence rainforest distribution and species diversity and evidence of a high degree of variability in terms of conservation. While some phylogenetically distinctive rainforest community types were well conserved, restricted or endangered communities were very poorly protected. Additionally, we found smaller dry rainforests in the Central Queensland Coast represent regional plant migration but are inadequately protected, highlighting the need for a revision of conservation objectives within the region.

In our recent examination of the Phintella specimens collected from Hainan Tropical Rainforest National Park, a new species and the unknown female of P. liae Wang, Mi & Peng, 2023 were recognised, based on the morphological characteristics and molecular evidence.A new species of Phintella Strand, 1906 is described: P. hongkan sp. nov. (♂♀) from Hainan, China. The unknown female of P. liae Wang, Mi & Peng, 2023 is also described for the first time. Diagnostic photos of both species are provided.

The genus Carnus Nitzsch, 1818 comprises small ectoparasites that feed on the blood of juvenile avians. They are characterised by dealated adults with setose abdominal intersegmental membranes. Carnus orientalis Maa, 1968 was previously recorded in Malaysia and the Ryukyu Islands of Japan, parasitising two owl species: Ketupa ketupu (Horsfield, 1821) and Otus elegans (Cassin, 1852). This study confirms the occurrence of C. orientalis in Taiwan and presents a new host record, along with COI barcode sequences. Additionally, the study also elucidates the difficulties posed by blood meal contamination and pseudogene amplification as confounding factors intrinsic to the molecular taxonomic delineation of C. orientalis via universal DNA barcoding primers.The following new information regarding C. orientalis is provided in this study:Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan.Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling.Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon.New specific primers for the COI gene of Carnus were designed in this study.The new distribution and ecological data of C. orientalis enhance our understanding of this species. The provision of new COI primers is anticipated to contribute to future studies employing DNA barcoding in bird-parasitic flies.

Thoracophelia Ehlers, 1897 is a genus of Opheliidae characterised by the body divided into three distinct regions, modified parapodia in chaetiger 10 and a ventral groove restricted to the posterior half of the body. To date, 18 species have been described in the genus. Amongst them, six species have been recorded in northeast Asia.A new species, Thoracophelia foliformis sp. nov., was discovered in the intertidal zone of the Yellow Sea, South Korea. This is the first Thoracophelia species report from the Yellow Sea. This new species is closely related to T. dillonensis (Hartman, 1938) from California and T. ezoensis Okuda, 1936 from Japan in having pectinate branchiae. However, the new species can be distinguished from the two species by the unique combination of the following characteristics: 15 pairs of wrinkled pectinate branchiae with 12–15 filaments at best development and a foliaceous mid-ventral plate in the pygidium instead of one or two thick ventral cirri. Detailed descriptions and illustrations of T. foliformis sp. nov. are provided. Sequences of the mitochondrial cytochrome c oxidase subunit I (COI), nuclear 18S ribosomal DNA (rDNA) and 28S rDNA of the new species were determined and analysed.

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The genus Acronyctodes Edwards is reexamined following caterpillar rearing and natural history observations, examination of genitalia, and DNA (COI) barcode analyses. Adults, caterpillars, and genitalia of known Mexican Acronyctodes are described and illustrated, and detailed distribution maps primarily derived from iNaturalist data are presented. Acronyctodes eximia (Hoffman & Vázquez) syn. n. is synonymized with A. cautama (Schaus), following a thorough examination of adult morphology, life history data, and DNA barcode analyses. The analysis of 128 COI barcodes, 18 of these representing unique haplotypes from five of the six described species, reveals that three can be reliably distinguished with this fragment. DNA barcodes support two species groups previously suggested by morphology: A. leonilaria + A. mexicanaria and A. cautama + A. colorata. New host plant records demonstrate Acronyctodes species are oligophagous and the study of distributional records reveal A. mexicanaria is sympatric to both A. cautama and A. leonilaria in parts of their ranges. [ABSTRACT FROM AUTHOR]

Holoplanktonic organisms are thought to have enormous populations sizes, expansive geographic ranges, and low species diversity. Previous work suggests that pelagic gastropods are no exception to this generalization. However, most of these data are derived from temperate species, whereas the bulk of pteropod diversity occurs in the tropics. Here, we present DNA barcode data for COI focused on the under‐studied limacinoid, gymnosome, and pseudothecosome pteropods collected from the coastal waters of Panama. We applied four molecular species delimitation approaches to determine the number of operational taxonomic units (OTUs) identifiable from our sequences and all data available from Barcode of Life Database (BOLD) and GenBank and compared these to the Barcode Index Number generated by the BOLD. Assemble Species by Automatic Partitioning (ASAP), Poisson Tree Processes (PTP), Bayesian Poisson Tree Processes (bPTP), and Generalized Mixed Yule Coalescent (GMYC) methods generally gave congruent results and suggest that pteropod diversity in the tropics is significantly underrepresented both in DNA sequence databases and in the number of named OTUs. Of 42 specimens collected and sequenced, we recovered 16 OTUs, only one of which belonged to an OTU already represented in sequence databases. Neighbor‐joining trees including the previously published sequences show that in all three groups morphospecies can include genetically divergent lineages. Many very divergent (>15% from nearest neighbor) taxa are also still represented by only a single sequence, suggesting that there is a large amount of cryptic or pseudo‐cryptic diversity still to be described. To aid this future endeavor, we include some preliminary 16S data derived from new pteropod‐specific primers. [ABSTRACT FROM AUTHOR]

Background: Culex tritaeniorhynchus has long been considered the primary vector of Japanese encephalitis virus (JEV), but until recently, it was considered exotic to Australia. When the species was detected in the country's Northern Territory (NT) for the first time, the Western Australia (WA) Department of Health was cognisant of the risk it posed to the State because of the shared border and continuous mosquito habitat adjoining the two jurisdictions. The aim of this study was to undertake intensive mosquito surveillance in the Kimberley region to ascertain whether Cx. tritaeniorhynchus was present in WA, define the extent of its distribution and undertake phylogenetic analysis of select specimens to support hypothesized routes of entry into the state. Methods: Carbon dioxide (CO2)-baited encephalitis virus surveillance (EVS) mosquito traps were deployed at various sites throughout the Kimberley region by surveillance officers within the Medical Entomology unit of the Western Australia (WA) Department of Health. Mosquitoes were then morphologically identified, and a subset of four specimens were confirmed as Cx. tritaeniorhynchus by molecular identification using Cytochrome Oxidase I (COI) DNA data and phylogenetic analysis. Results: From 31 March 2021 to 30 May 2024, a total of 211 female Cx. tritaeniorhynchus specimens were collected from 21 unique trap sites in the Kimberley's Shire of Wyndham-East Kimberley (SWEK). Four COI DNA barcode regions were amplified and successfully sequenced for analysis. These sequences fell within a clade recognised as Cx. tritaeniorhynchus and specifically all sequences were in a clade with other specimens from the NT and Timor-Leste. Conclusions: This study represents the first detection of Cx. tritaeniorhynchus in WA. Given the widespread nature of trap sites that yielded the species and consecutive seasons over which it was observed, the authors surmise that Cx. tritaeniorhynchus is now established within the northeast Kimberley region. The findings are significant given the detection of the species coincides with the first significant outbreak of JEV activity on mainland Australia involving an estimated 45 human cases of Japanese encephalitis, 80 impacted commercial piggeries and widespread feral pig activity. Although the role that Cx. tritaeniorhynchus may play in JEV transmission into the future is not yet understood, it presents a potential risk to public health in the region. [ABSTRACT FROM AUTHOR]

Dry rainforest communities are globally threatened by anthropogenic pressures and climatic change but are less well researched and more poorly conserved than mesic rainforests. In response to the increasing loss of biodiversity, the Australian Government joined other international signatory parties to adopt the Kunming-Montreal Global Biodiversity Framework (GBF). The GBF emphasises the maintenance of connectivity and genetic diversity of whole ecosystems via landscape-scale conservation initiatives. Rainforest plant diversity, distinctiveness, and the current level of conservation of seasonal rainforest regional ecosystems of the Central Queensland Coast region in Australia were evaluated. Our three-marker DNA barcode dated phylogeny of rainforest plant taxa together with community species lists were used to calculate phylogenetic diversity (PD) estimates and species composition. Levels of rainforest ecosystem protection were assessed using Queensland government data. This study found selection pressures for moisture and geology significantly influence rainforest distribution and species diversity and evidence of a high degree of variability in terms of conservation. While some phylogenetically distinctive rainforest community types were well conserved, restricted or endangered communities were very poorly protected. Additionally, we found smaller dry rainforests in the Central Queensland Coast represent regional plant migration but are inadequately protected, highlighting the need for a revision of conservation objectives within the region. [ABSTRACT FROM AUTHOR]

Genetic diversity and ecology are two important aspects of vector species crucial to a full understanding of disease epidemiology. In this study, we examined genetic diversity, genetic structure and the physiochemical parameters of the breeding habitats of the two significant black fly vector species, Simulium chumpornense Takaoka and Kuvangkadilok and S. nodosum Puri, from Laos. Genetic diversity of S. chumpornense in Laos was relatively high with maximum genetic divergence of 3.05% but no significant genetic differentiation between populations. Comparisons with conspecific populations from Thailand also found no genetic differentiation between the two countries. This possibly related to the recent history of this species, as a very recent (31,000 years ago) historical population expansion was detected. Physicochemical parameters of the breeding habitats suggest the ability to utilize diverse stream sizes from small flows (1 m wide) to huge rivers (290 m or more wide). Populations of S. nodosum from Laos had low genetic diversity with maximum genetic divergence of 2.56% and no genetic structuring among populations. Comparisons with those reported from other countries revealed five genetically divergent lineages (I–V) with minimum genetic divergence of 1.36%. The majority (42 of 52) of specimens from Laos belonged to lineage I and the remaining (10) comprised lineage II. Lineage I is the largest and representative of specimens from Thailand, Myanmar, Vietnam, Taiwan and Laos. Population history analysis revealed that lineage I had undergone recent demographic expansion dating back to 7000 years ago. This very recent population expansion resembles others reported from Thailand and possibly indicates a response to increasing human and domestic animals following the agricultural revolution. Breeding sites of S. nodosum are diverse in respect to elevation, velocity, water conductivity and streambed particle size. The ability to utilize a wide variety of breeding sites could promote the production of a large adult population, which can be a pest for humans and other animals. [ABSTRACT FROM AUTHOR]

Astylus atromaculatus Blanchard, 1843 (Coleoptera: Melyridae) is a pollinivorous beetle native to southern South America, which has invaded South Africa more than a century ago. Adults and/or larvae may occasionally damage flowers, seeds, and seedlings of various crops. Severe cattle intoxication has also been reported in Argentina, Uruguay, and South Africa following consumption of alfalfa and forage grasses infested with A. atromaculatus. Despite its economic impact, essential genetic information is lacking for this species. The present paper provides the first DNA barcode reference sequences for A. atromaculatus based on the standard 5’ fragment (658 bp) of the cytochrome c oxidase subunit I gene. The sequences obtained exhibited pairwise distances of ≤ 1.82% among them, and ~ 90% nucleotide identity with the homologous gene fragment in the morphologically similar Astylus variegatus Germar, 1824. The use of this molecular marker to explore the intraspecific variability of A. atromaculatus in central Argentina showed 21 different haplotypes, out of 32 individuals analyzed. A very high haplotype diversity (Hd = 0.962 ± 0.019) and a moderate nucleotide diversity (π = 0.00778 ± 0.00079) were recorded. The haplotype network displayed a diffuse structure due to the abundance of singletons and possible missing haplotypes, with the most common haplotype comprising only 15.6% of the specimens collected. Future research with increased sampling size and geographic coverage will allow for a better understanding of the population genetics of this pest, and consequently, for developing efficient management practices. [ABSTRACT FROM AUTHOR]

Simple Summary: The tribe Dacnusini is a small braconid parasitoid wasp within the subfamily Alysiinae (Hymenoptera: Braconidae), consisting of 31 valid genera and more than 877 species worldwide. Most members are exclusively endoparasitoids of leaf-mining Diptera (Agromyzidae), and several species have been utilized in commercial biological control programs, underscoring their essential role in natural pest management. However, due to low interspecific variability and significant morphological convergence, distinguishing between genera and species within the genera in Dacnusini presents a challenge. In this study, we report three genera, Victorovita Tobias, Coloneura Foerster, and Laotris Nixon, that were discovered in China for the first time. By integrating DNA barcoding and morphological evidence, three new species were identified through multiple species delimitation methods. Additionally, two new species records from China are reported, and identification keys for species of Laotris Nixon, 1943 are provided. Dacnusini is a species-rich tribe in the subfamily Alysiinae, with most species exclusively serving as parasitoids of leaf-mining Diptera (Agromyzidae). The number of genera discovered in China remains limited, which is apparently insufficient considering the global diversity of species and genera within this tribe, particularly given the vast and ecologically diverse landscapes of China. In the present study, three new record genera, Victorovita Tobias, Coloneura Foerster, and Laotris Nixon, were documented for the first time in China. In addition, the species delimitation approach and haplotype network analyses based on the COI sequences, combined with morphological evidence, were employed to delimit species. The findings indicated three new species: Laotris glabella sp. nov., Laotris aethidentata sp. nov., and Victorovita aequalis sp. nov. Additionally, K2P divergences showed no overlap between intra- and interspecific genetic distances in the Laotris and Victorovita species. Detailed descriptions for new species and keys to the species of Laotris and Victorovita are provided in this paper, along with the documentation of two new species records for China: Victorovita caudata (Szépligeti, 1901) and Coloneura stylata Foerster, 1863. [ABSTRACT FROM AUTHOR]

Background: The genus Carnus Nitzsch, 1818 comprises small ectoparasites that feed on the blood of juvenile avians. They are characterised by dealated adults with setose abdominal intersegmental membranes. Carnus orientalis Maa, 1968 was previously recorded in Malaysia and the Ryukyu Islands of Japan, parasitising two owl species: Ketupa ketupu (Horsfield, 1821) and Otus elegans (Cassin, 1852). This study confirms the occurrence of C. orientalis in Taiwan and presents a new host record, along with COI barcode sequences. Additionally, the study also elucidates the difficulties posed by blood meal contamination and pseudogene amplification as confounding factors intrinsic to the molecular taxonomic delineation of C. orientalis via universal DNA barcoding primers. New information: The following new information regarding C. orientalis is provided in this study: Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan. Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling. Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon. New specific primers for the COI gene of Carnus were designed in this study. The new distribution and ecological data of C. orientalis enhance our understanding of this species. The provision of new COI primers is anticipated to contribute to future studies employing DNA barcoding in bird-parasitic flies. [ABSTRACT FROM AUTHOR]

Background: In our recent examination of the Phintella specimens collected from Hainan Tropical Rainforest National Park, a new species and the unknown female of P. liae Wang, Mi & Peng, 2023 were recognised, based on the morphological characteristics and molecular evidence. New information: A new species of Phintella Strand, 1906 is described: P. hongkan sp. nov. (♂♀) from Hainan, China. The unknown female of P. liae Wang, Mi & Peng, 2023 is also described for the first time. Diagnostic photos of both species are provided. [ABSTRACT FROM AUTHOR]

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

Background: Thoracophelia Ehlers, 1897 is a genus of Opheliidae characterised by the body divided into three distinct regions, modified parapodia in chaetiger 10 and a ventral groove restricted to the posterior half of the body. To date, 18 species have been described in the genus. Amongst them, six species have been recorded in northeast Asia. New information: A new species, Thoracophelia foliformis sp. nov., was discovered in the intertidal zone of the Yellow Sea, South Korea. This is the first Thoracophelia species report from the Yellow Sea. This new species is closely related to T. dillonensis (Hartman, 1938) from California and T. ezoensis Okuda, 1936 from Japan in having pectinate branchiae. However, the new species can be distinguished from the two species by the unique combination of the following characteristics: 15 pairs of wrinkled pectinate branchiae with 12–15 filaments at best development and a foliaceous mid-ventral plate in the pygidium instead of one or two thick ventral cirri. Detailed descriptions and illustrations of T. foliformis sp. nov. are provided. Sequences of the mitochondrial cytochrome c oxidase subunit I (COI), nuclear 18S ribosomal DNA (rDNA) and 28S rDNA of the new species were determined and analysed. [ABSTRACT FROM AUTHOR]

Euphorbiae pekinensis Radix (EPR) is a traditional Chinese herb commonly used to treat edema, pleural effusion, and ascites. However, counterfeit and adulterated products often appear in the market because of the homonym phenomenon, similar appearance, and artificial forgery of Chinese herbs. This study comprehensively evaluated the quality of EPR using multiple methods. The DNA barcode technique was used to identify EPR, while the UPLC‐Q‐TOF‐MS technique was utilized to analyze the chemical composition of EPR. A total of 15 tannin and phenolic acid components were identified. Furthermore, UPLC fingerprints of EPR and its common counterfeit products were established, and unsupervised and supervised pattern recognition models were developed using these fingerprints. The backpropagation artificial neural network and counter‐propagation artificial neural network models accurately identified counterfeit and adulterated products, with a counterfeit ratio of more than 25%. Finally, the contents of the chemical markers 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐D‐glucopyranoside, ellagic acid, 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐d‐xylopyranoside, and 3,3′‐di‐O‐methyl ellagic acid were determined to range from 0.05% to 0.11%, 1.95% to 8.52%, 0.27% to 0.86%, and 0.10% to 0.42%, respectively. This proposed strategy offers a general procedure for identifying Chinese herbs and distinguishing between counterfeit and adulterated products. [ABSTRACT FROM AUTHOR]

Cassia species are considered as a valuable source for our Indian medical system. Inter and intra species of Cassia is genetically variable and its necessary to identify and discriminate individuals in order to exploit its maximum potential. DNA barcode technique of well-studied universal markers was used to amplify the genomic region of several Cassia species. The amplified regions were sequenced and the coding's were blasted and studied for any significant differences. The results from the generated Rbcl, MatK and Ycf1b sequences were almost identical to Senna species that is closely related to Cassia species. The phylogenetic tree for five species of Cassia was constructed using three markers (Rbcl, MatK and Ycf1b). Among the markers used Rbcl_1 shows the best resolution for S. auriculata. MatK_3 has showed better species resolution for S. occidentails than other markers. The implication of these results can be used in species identification of wild and native genotypes of medicinal plant from various geographical locations. [ABSTRACT FROM AUTHOR]

Abstract Background Black flies are among the most medically and veterinary important insects, as adult females of certain species are the sole vector of Onchocerca volvulus. Here, a new black fly species belonging to the subgenus Asiosimulium Takaoka & Choochote, 2005, is described and formally named as Simulium (Asiosimulium) kittipati sp. nov. Methods Pupae and larvae of black flies were collected from available substrates in the stream from central Thailand. Pupae were individually separated in plastic tubes and maintained until adult flies emerged. The emerged adult flies associated with their pupal exuviae and cocoon as well as mature larvae preserved in 85% ethanol were used to describe the new species based on an integrated approach of morphological examination and molecular analysis of the COI gene. Results The new species is characterized in the female by the medium-long sensory vesicle with a medium-sized opening apically, scutum with three faint longitudinal vittae, and the ellipsoidal spermatheca; in the male by the number of upper-eye (large) facets in 20 vertical columns and 21 horizontal rows, hind basitarsus slender, nearly parallel-sided, and median sclerite much wider and upturned apically; in the pupa by the head and thoracic integument densely covered with tiny tubercles, and the pupal gill of arborescent type with 28–30 filaments; and in the larva by the postgenal cleft deep, nearly reaching the posterior margin of the hypostoma, and dark pigmented sheath of the subesophageal ganglion. The DNA barcode successfully differentiated the new species from its congeners with an interspecific genetic divergence of 1.74–18.72%, confirming the morphological identification that the species is a new member of the subgenus Asiosimulium. Phylogenetic analyses also indicated that the new species is genetically closely related to Simulium phurueaense Tangkawanit, Wongpakam & Pramual, 2018, further supporting its morphological classification. Conclusions This is the ninth species assigned to the subgenus Asiosimulium within the genus Simulium Latreille, 1802. Taxonomic notes and identification keys are given to distinguish this new species from the eight known species members in its same subgenus. Additionally, a distribution map of all species members in this subgenus occurring in Thailand and other countries is provided. Graphical abstract

DNA barcodes, short and unique DNA sequences, play a crucial role in sample identification when processing many samples simultaneously, which helps reduce experimental costs. Nevertheless, the low quality of long-read sequencing makes it difficult to identify barcodes accurately, which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run. Here, we present a comprehensive study of the generation of barcodes and develop a tool, PRO, that can be used for selecting optimal barcode sets and demultiplexing. We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete. For practical applications, we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy. Specifically, the maximum size of the barcode kits designed by PRO is 2,292, which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies (ONT). We validated the performance of PRO on a simulated nanopore dataset with high error rates. The demultiplexing accuracy of PRO reached 98.29% for a barcode kit of size 2,922, 4.31% higher than that of Guppy, the official demultiplexing tool. When the size of the barcode kit generated by PRO is the same as the official size provided by ONT, both tools show superior and comparable demultiplexing accuracy.

INTRODUCTION. The creation of synthetic adeno-associated virus (AAV) vectors during gene therapy development is a labour-intensive and expensive process. The optimal solution to minimise the time and costs associated with gene therapy development lies in the improvement of methods aimed at assessing AAV vector biodistribution and transduction efficiency in vivo.AIM. This study aimed to develop a new bioinformatics-based assessment method for synthetic AAV vector libraries to analyse AAV vector biodistribution and transduction efficiency in vivo.MATERIALS AND METHODS. The production of synthetic AAV vectors involved assigning AAV serotype-specific barcodes (12-nucleotide tags flanked at the 5' end with a sequence encoding the green fluorescent reporter protein). Plasmids carrying unique barcodes were propagated in competent Escherichia coli XL10-Gold cells and used to create two AAV libraries: L1 with a viral genome count of 1010 and L2 with a viral genome count of 1011. AAV production involved HEK293T cell transfection. L1 and L2 library vectors were administered to C57Bl/6N mice by intravenous injection. DNA and RNA were isolated from transduced organs for analysis by next-generation sequencing. The obtained data on DNA and RNA barcode quantities in different murine organs were analysed to assess the biodistribution and transduction efficiency of synthetic AAVs. Barcodes were identified by aligning them to the expected sequences and counted. The resulting values were normalised to the quantity of barcodes in the original library.RESULTS. Seven viral constructs based on different AAV serotypes were created as part of two AAV libraries. Six of the AAV serotypes were synthetic (sAAV1, sAAV2, sAAV3, sAAV4, sAAV5, and sAAV6). Sequencing of murine organ samples revealed significant quantities of DNA barcodes from both AAV libraries in all organs except the brain. For the L1 library, RNA barcodes were detected at a sufficient level in 4 organs, including the skeletal muscles, the heart, the liver, and the adrenal glands. For the L2 library, in addition to the listed organs, sufficient RNA-barcode levels were observed in the gonads and the kidneys. According to transduction efficiency analysis based on RNA barcode levels adjusted for DNA barcodes, sAAV5 was considered the most promising variant for gene therapy of liver-related diseases, whereas sAAV2 and sAAV6 were recognised as holding the most promise for adrenal diseases.CONCLUSIONS. The developed bioinformatics-based assessment method for synthetic AAV vector libraries can analyse AAV vector biodistribution and transduction efficiency in the body. The presented approach has the potential for selecting optimal AAV vectors for specific organs and tissues in further gene therapy development.

Background: Black flies are among the most medically and veterinary important insects, as adult females of certain species are the sole vector of Onchocerca volvulus. Here, a new black fly species belonging to the subgenus Asiosimulium Takaoka & Choochote, 2005, is described and formally named as Simulium (Asiosimulium) kittipati sp. nov. Methods: Pupae and larvae of black flies were collected from available substrates in the stream from central Thailand. Pupae were individually separated in plastic tubes and maintained until adult flies emerged. The emerged adult flies associated with their pupal exuviae and cocoon as well as mature larvae preserved in 85% ethanol were used to describe the new species based on an integrated approach of morphological examination and molecular analysis of the COI gene. Results: The new species is characterized in the female by the medium-long sensory vesicle with a medium-sized opening apically, scutum with three faint longitudinal vittae, and the ellipsoidal spermatheca; in the male by the number of upper-eye (large) facets in 20 vertical columns and 21 horizontal rows, hind basitarsus slender, nearly parallel-sided, and median sclerite much wider and upturned apically; in the pupa by the head and thoracic integument densely covered with tiny tubercles, and the pupal gill of arborescent type with 28–30 filaments; and in the larva by the postgenal cleft deep, nearly reaching the posterior margin of the hypostoma, and dark pigmented sheath of the subesophageal ganglion. The DNA barcode successfully differentiated the new species from its congeners with an interspecific genetic divergence of 1.74–18.72%, confirming the morphological identification that the species is a new member of the subgenus Asiosimulium. Phylogenetic analyses also indicated that the new species is genetically closely related to Simulium phurueaense Tangkawanit, Wongpakam & Pramual, 2018, further supporting its morphological classification. Conclusions: This is the ninth species assigned to the subgenus Asiosimulium within the genus Simulium Latreille, 1802. Taxonomic notes and identification keys are given to distinguish this new species from the eight known species members in its same subgenus. Additionally, a distribution map of all species members in this subgenus occurring in Thailand and other countries is provided. [ABSTRACT FROM AUTHOR]

The jumping spider species Gedea pinguis Cao & Li, 2016 is newly recorded from Vietnam and redescribed based on the sole male specimen. A detailed redescription, illustrations of the copulatory organs, and somatic features, along with an updated distribution map are provided. DNA barcode derived from the mitochondrial cytochrome c oxidase subunit I (COI) gene of the species G. pinguis from Vietnam is also included for future use and to compare the genetic distances with Gedea cf. tibialis Zabka, 1985 from Malaysia. With the addition of a new record, the genus Gedea Simon, 1902 in Vietnam now reaches 3 species, but doubts arise regarding the conspecificity of the two opposite sex species, Gedea tibialis Żabka, 1985 (male) and Gedea typica (Żabka, 1985) (female), as they share the same type locality. Additionally, a dichotomous key to all Gedea species is also given in this study. [ABSTRACT FROM AUTHOR]

The Afrotropical Cerurinae (Notodontidae) genera Cerurina Kiriakoff and Afrocerura Kiriakoff are revised. Historically, significant confusion has existed surrounding the identities of members of these 2 closely related genera. Using morphology and genetic data derived from mitochondrial COI barcodes and anchored hybrid enrichment nuclear genomics, we present an integrated taxonomic approach. Upon examining all types, historical material in global collections, and numerous recently collected specimens, we are able to associate available names with the taxa in these genera as well as describe four new species of Afrocerura for which no names were available. Historical misidentifications are also addressed. For nearly all species, both sexes and their genitalia are figured. In total, we recognize one species in Cerurina with 2 subspecies: C. marshalli marshalli Hampson (type species) and C. marshalli cameroona (Bethune-Baker) comb. n. , and 8 species in Afrocerura : A. leonensis (Hampson) (type species), A. takanoi sp. n. , A. bifasciata (Janse), A. inexpectata sp. n. , A. tanganyikae Kiriakoff stat. n. , A. ethiopica sp. n. , A. thomensis (Talbot) and A. smithi sp. n. Color photos of C. m. cameroona larvae are presented for the first time and are compared to larvae of C. m. marshalli. [ABSTRACT FROM AUTHOR]