Considering that bat ecology alterations may be linked with pathogen spillover, research on bat coronaviruses, particularly on the infection and transmission pattern among bats in relation with their ecology, is essential. We captured bats distributed in Korea from 2021 to 2022, examined coronaviruses in oral swabs, feces, urine, and ectoparasites, and were able to detect alphacoronavirus. We investigated coronaviruses, but noted no substantial differences in the body condition index in the coronavirus-positive bats. Binary logistic regression analysis revealed that bat ecological factors that were significantly associated with coronavirus-positive were roost type, sample type, and bat species. Coronavirus-positive ectoparasite cases suggested additional study on the potential role of them as the viral transmission vectors or fomites. Reinfection of a different coronavirus in recaptured bats was evident, suggesting the possibility that coronavirus circulation can evade the potential protective immunity acquired from previous coronavirus infections. The present findings provide comprehensive information on the coronaviruses transmission dynamics within bat populations linked with bat ecology.

Genus Ceratomyxa comprises coelozoic parasites of mainly marine and brackish water fish. This study describes a new Ceratomyxa species, Ceratomyxa schwanefeldii n. sp. which parasitizes the gall bladder of Barbonymus schwanefeldii collected from Sungai Tong in Setiu, Terengganu, Malaysia. The new species was described using morphological characteristics, and on nucleotide sequences of small subunit ribosomal DNA (SSU rDNA) and large subunit ribosomal DNA (LSU rDNA). Ceratomyxa schwanefeldii n. sp. exhibited vermiform shape plasmodia with slow undulatory motility, measuring 151.6 ± 86.0 (43.0-271.0) μm in length and 15.1 ± 4.8 (9.3-22.7) μm in width, with blunt poles at both ends. The mature spores were crescent-shaped, strongly arched in frontal view, with a sutural line between the two valves tapering to blunt ends. Formalin-fixed spores were 3.0 ± 0.4 (2.4-3.9) μm in length, 12.6 ± 1.2 (10.8-15.4) μm in thickness, with a concave posterior angle, 104.8° ± 10.2° (73.4-123.8). Two equal-sized spheroid polar capsules measured 1.5 ± 0.2 (1.2-1.8) μm in length and 1.3 ± 0.2 (0.9-1.7) μm in width. Phylogenetic analyses by Maximum likelihood and Bayesian Inference algorithms positioned C. schwanefeldii n. sp. as a sister species to Unicapsulocaudum mugilum and clustered within the clade of Amazonian freshwater Ceratomyxa species. The LSU rDNA phylogeny revealed that C. schwanefeldii n. sp. clusters within the marine Ceratomyxa clade and forms a sister relationship with C. leatherjacketi. This study represents the first description of a freshwater Ceratomyxa in Malaysia and the fourth recorded detection in the Asian region.

Elaeophora (Nematoda: Onchocercidae), a filarial nematode infecting the blood vessels of ruminants and horses, is transmitted by tabanid flies. Elaeophora elaphi was previously detected in wild sika deer in Wakayama Prefecture, Japan in 2009; however, detailed information on this species is scarce. In 2023, 26 Elaeophora worms were collected from the hepatic vasculature of eight deer in Nara, Mie, Kyoto and Gifu Prefectures of Japan and analyzed. Species identification was performed by morphological and genetic analyses. Additionally, multi-gene analysis of seven genes was performed to determine their taxonomic position within the family Onchocercidae. The specimens were identified as E. elaphi based on their morphological characteristics. Analyses of 18S rRNA and cytochrome c oxidase subunit 1 genes revealed no variations, indicating that species belonged to the same lineage. Multi-gene analysis revealed that the species belonged to the subfamily Onchocercinae, showing a close relationship with the tick-borne filarial nematodes of the genera Monanema, Acanthocheilonema, Litomosoides, Cruorifilaria, Yatesia, and Cercopithifilaria. This study demonstrated the widespread distribution of E. elaphi in Japan and provided insights into its genetic relationship with other onchocercid species. Further research is necessary to determine the ecological and epidemiological implications of this parasite.

Microplastics (MPs) have wreaked havoc on aquatic ecosystems due to their heightened accumulation potential. Polyethylene (PE) is the most dominant MP polymer contaminating several estuaries globally. This study aims to evaluate the acute exposure of MPs on two size classes of mangrove horn snail, Telescopium telescopium. Small (50-70 mm) and large (90-120 mm) sized organisms were exposed to polyethylene microplastics (PE-MPs) of diameter 34-50 μm, for a period of 21 days. PE-MP toxicity was assessed by monitoring the physiological energetics and oxidative stress biomarker responses at three environmentally relevant concentrations (2, 20, 200 μg L-1). Negative Scope for Growth was observed in both size classes at medium and high concentrations. Moreover, PE-MPs significantly induced oxidative damage at 20 and 200 μg L-1 concentrations. Noticeable DNA damage was observed across all PE-MP concentrations. Small sized gastropods were more vulnerable to MP toxicity than their larger counterparts. Integrated Biomarker Response indicated Superoxide Dismutase as the most responsive biomarker for assessing the toxicological effects of MPs. Present study provides new insights on the potential impacts of PE-MPs on different size classes of T. telescopium from Sundarbans Estuarine System.

To investigate the biological effects of the ABA receptor pyrabactin resistance 1-like (PYR1-like) in Malus sieversii seeds, the proteins interacting with MsPYR1-like were screened by the membrane yeast two-hybrid library based on the split-ubiquitin system, and to construct the bait vector pBT3-SUC-PYR1 for Malus sieversii cDNA library, which had no self-activating effect on the yeast cells of the pPR3-N membrane yeast two-hybrid library. The library titer assay showed that it could meet the requirements for membrane yeast two-hybrid library screening. After sequencing, GenBank database blast, and yeast rotary validation, 28 candidate proteins interacting with MsPYR1-like were obtained, including ribosomal proteins, late embryogenesis abundant proteins, F-actin-capping proteins, phytochrome-interacting proteins, low-temperature-inducible 65 kDa protein-like, senescence-associated, PP2C and SnRK2 family members, and unknown proteins. Gene ontology analysis of the interaction proteins was related to plant hormone response and negative regulation of seed germination, overexpression of MsPYR1-like in Arabidopsis negatively regulates seed germination, and the study of the biological roles of MsPYR1-like interacting proteins lays the foundation for revealing the lifting of seed dormancy in Malus sieversii.

Alternative oxidase (AOX) (EC 1.10.3.11) is a terminal oxidase in the mitochondrial inner membrane that branches the canonical electron transport system (ETS). AOX is ubiquitous in plants, frequently found in fungi and protists and presents a more sporadic distribution in metazoans. More recently, AOX has gained attention due to its potential application in gene therapy for treatment of mitochondrial diseases. Here we characterized the AOX in the basal Dipteran, Pseudolycoriella hygida using a combination of genomic analyses, molecular, functional and in vivo survival assays. AOX is a single copy gene that encodes three developmental stage specific protein isoforms. AOX localizes to the mitochondria in adult thoracic muscles, which present cyanide-resistant respiration that is sensitive to the AOX inhibitor salicylhydroxamic acid (SHAM). Both the cyanide-resistant respiration and AOX levels gradually increase during aging, but are not influenced by thermal stress. Thoracic mitochondria respire using substrates derived from several metabolic routes, such as pyruvate, proline, acylcarnitine, NADH and glycerol-3P, and present values of oxidative phosphorylation capacity ((P-L)/E = 0.70) and coupling (P/L = 4.35; L/E = 0.21). Adult flies exhibit a high survival resistance for SHAM-sensitive complex III inhibition. Together, our results demonstrate the presence of a functional AOX in a terrestrial arthropod and provide insights regarding AOX function in animals and evolution of respiratory systems in metazoans. Psl. hygida emerges as a natural and valuable model for comprehensive AOX research at the whole-organism level which complements models expressing the heterologous enzyme.

Mass mortalities with clinical signs characteristic of acute hepatopancreatic necrosis disease (AHPND) were reported in cultured Litopenaeus vannamei from three Egyptian farms: Wadi-Mariote, Berket Ghalyoun, and Qarun Lake. During 4-month surveillance in 2023, shrimp samples were collected to investigate the prevalence of AHPND-causing Vibrio parahaemolyticus (VpAHPND) based upon phenotypic identification, PCR, and DNA sequencing of pirA genes and pirB toxin genes followed by maximum likelihood phylogenetic analysis. In addition, the pathogenicity of V. parahaemolyticus was evaluated through the injection challenge and histopathological examination. Moreover, the antibacterial activity of marine fungal extracts against identified V. parahaemolyticus was also assessed. Molecular analysis confirmed both pirA (282 bp) and pirB (392 bp) toxin genes in the bacterial isolates. A significant positive correlation (P < 0.05) was found between V. parahaemolyticus levels in shrimp and pond water samples throughout the study period. Injection challenge with 2.7 × 104 CFU bacterial suspension resulted in 63.33 % mortality in challenged shrimp, with typical AHPND clinical signs. The histopathological examination revealed degenerative changes, including atrophy, necrosis, and sloughing of hepatopancreatic tubule epithelial cells, along with loss of functional hepatopancreatic cells. Among 11 fungal isolates screened for anti-vibrio activity, Aspergillus niger HMA9 showed the strongest inhibitory effect against V. parahaemolyticus. This study provides the first genetic confirmation of pirA and pirB toxin genes in VpAHPND as the cause of L. vannamei mass mortalities in Egypt. Further, it demonstrates the potential of fungal bioactive compounds for controlling AHPND in shrimp aquaculture.

A pan-genome includes the complete pool of genes of a species including those recently acquired. The new additions of genetic material to a genome are frequently linked to horizontal gene transfer (HGT) processes and can confer adaptive advantages improving the recipient functional response and growth. Previous studies have reported that Parageobacillus have frequent DNA exchange mainly with other members of the phylum Bacillota sharing similar environments. Nevertheless, the occurrence of transfer events between phylogenetically distant microorganisms is scarcely known. In this work, based on the pan-genome of Parageobacillus thermoglucosidasius, we detected a number of unique genes within the species which were used to carry out BLAST searches to find out similar genes in distant bacteria taxa. We aimed to infer potential inter-phylum HGT events. Results suggested genetic exchanges among different phyla. Among them Actinomycetota, Pseudomonadota and the Bacteroidota/Chlorobiota group were the dominant observed phyla. Those HGT events frequently involved ATP binding cassette transporters, enzymes of the C metabolism and transcriptional regulators. Based on the frequency of these genes within specific phyla, directional HGT events could be proposed. A dominant origin of the suggested HGT events could be within the Bacillota. This exploratory analysis indicates that Bacillota are frequent exporters of DNA both within the phylum and to phylogenetically distant groups. Long-distance HGT can assist to better understand microbial evolution, the relevance of HGT processes within the prokaryotes and the genomic plasticity of microorganisms.

Leptospira is a bacterial genus which includes several pathogenic species. Wild mammals can act as reservoir hosts, shedding bacteria in their urine. Leptospirosis is an important health problem in Villeta, but data regarding potential reservoirs hosts and natural sources of infection are still scarce. We aimed to detect and characterize the presence of Leptospira among small wild mammals from Villeta municipality, Colombia. Small wild mammals from three orders: Didelphimorphia, Chiroptera and Rodentia, were sampled in the region. DNA was extracted from kidney samples and screened for Leptospira through real-time PCR targeting a the 16 s rRNA gene. Positive samples were screened through conventional PCR using five complementary genes: adk, icdA, lipL32, lipL41 and secY. Amplicons were sequenced and used for phylogenetic analysis. A total of 75 kidney samples were collected from three small wild mammal orders: 62 bats, 5 opossums, and 7 rodents. Leptospira spp. was detected in 38.7 % of the samples, with bats presenting the most frequent infection rate (43.5 %). The infection rate varied by sampling site, with the highest frequency observed in Mave village (57.1 %). Concatenated phylogenetic analysis revealed that all sequences clustered within the P1/Pathogenic Leptospira major clade, forming three subclades: two bat-related Leptospira subclades, and one subclade including Leptospira interrogans, Leptospira kirschneri, and Leptospira noguchii species. This study describes the presence of Leptospira among bats and opossums from Villeta, Colombia. It also identifies the circulation of several P1/pathogenic Leptospira species among bats forming three clusters, two of them composed exclusively of bat-related leptospires, and one together with recognized pathogenic species.

Human mitochondrial DNA (mtDNA) harbors essential mutations linked to aging, neurodegenerative diseases, and complex muscle disorders. Due to its uniparental and haploid inheritance, mtDNA captures matrilineal evolutionary trajectories, playing a crucial role in population and medical genetics. However, critical questions about the genomic diversity patterns, inheritance models, and evolutionary and medical functions of mtDNA remain unresolved or underexplored, particularly in the transition from traditional genotyping to large-scale genomic analyses. This review summarizes recent advancements in data-driven genomic research and technological innovations that address these questions and clarify the biological impact of nuclear-mitochondrial segments (NUMTs) and mtDNA variants on human health, disease, and evolution. We propose a streamlined pipeline to comprehensively identify mtDNA and NUMT genomic diversity using advanced sequencing and computational technologies. Haplotype-resolved mtDNA sequencing and assembly can distinguish authentic mtDNA variants from NUMTs, reduce diagnostic inaccuracies, and provide clearer insights into heteroplasmy patterns and the authenticity of paternal inheritance. This review emphasizes the need for integrative multi-omics approaches and emerging long-read sequencing technologies to gain new insights into mutation mechanisms, the influence of heteroplasmy and paternal inheritance on mtDNA diversity and disease susceptibility, and the detailed functions of NUMTs.

Roflumilast, the third phosphodiesterase-IV (PDE4) inhibitor approved for use in dermatology, is indicated for topical treatment of psoriasis, seborrheic dermatitis, and atopic dermatitis, whereas its 2 predecessors, apremilast and crisaborole, are indicated for oral treatment of psoriasis and topical treatment of atopic dermatitis, respectively. All 3 are rationally designed PDE4 inhibitors, but roflumilast is the most potent and effective among the 3, with in vitro inhibitory constant half-maximal inhibitory concentration value of 0.7 nM (roflumilast), 0.14 μM (apremilast), and 0.24 μM (crisaborole), representing differences of over 3 orders of magnitude. PDE4 is a cAMP (an intracellular secondary messenger) hydrolase consisting of at least 4 subtypes of exon-spliced isoforms, which are primarily expressed in immune cells for inflammatory response. PDE4 inhibition lengthens the duration of cAMP signals and increases cellular cAMP concentrations, generating anti-inflammatory effects. We examined the physicochemical principles that make PDE4 inhibitors effective and propose chemical modifications to improve them. Sequence alignment of the catalytic domains of all phosphodiesterases identified many previously unreported invariant residues. These residues bind 1 Zn and 1 Mg ion plus 5 structural water molecules for orienting an attacking μ-hydroxyl/μ-oxo anion and for stabilizing 2 nonbridging phosphate oxygen atoms. The arrangement of the 2 divalent metal ions in phosphodiesterases is not related to that of the classic mechanism for general phosphoryl transfer.

Spodoptera frugiperda (fall armyworm) poses a substantial risk to crops worldwide, resulting in considerable economic damage. The gut microbiota of insects plays crucial roles in digestion, nutrition, immunity, growth and, sometimes, the degradation of insecticides. The current study examines the effect of synthetic insecticides on the gut microbiome of third instar S. frugiperda larvae using both culture-dependent techniques and 16S rRNA gene sequencing for bacterial community profiling and diversity analysis. In untreated larvae, the sequencing approach revealed a diverse microbiome dominated by the phyla Firmicutes, Proteobacteria and Bacteroidota, with key genera including Bacteroides, Faecalibacterium and Pelomonas. In parallel, 323 bacterial strains were isolated and assigned to the orders Bacillales, Burkholderiales, Enterobacterales, Flavobacteriales, Lactobacillales, Micrococcales, Neisseriaies, Pseudomonadales, Sphingobacteriales and Xanthomonadales. The prevailing culturable species included Serratia marcescens, Klebsiella variicola and Enterobacter quasiroggenkampii. Treatment with sublethal concentrations of three insecticides (broflanilide, spinosad and indoxacarb) caused significant changes in gut microbiome diversity and composition. Treated larvae showed a shift towards increased Proteobacteria abundance and decreased Firmicutes. Specifically, Acinetobacter and Rhodococcus were dominant in treated samples. Functional predictions highlighted significant metabolic versatility involving nutrient processing, immune response, detoxification, xenobiotic metabolism, and stress response, suggesting microbial adaptation to insecticide exposure. Network correlation analysis highlighted disrupted microbial interactions and altered community structures under insecticide treatment. These findings enhance our understanding of how insecticides impact the gut microbiota in S. frugiperda and may inform future strategies for managing pest resistance through microbiome-based approaches.

Fowl adenovirus (FAdV) poses incessant outbreaks to poultry production worldwide, and Inclusion body hepatitis (IBH) is a predominant FAdV infectious disease. Currently, limited vaccines are available in Malaysia to fight against the local predominant FAdV strain 8b isolate (FAdV-8b), posing a desperate demand for efficient vaccine development. The fiber protein of FAdV is one of the major constituents of the adenoviral capsid involved in the virulence of pathogens. Hence, the aim was to modify the fiber gene of FAdV-8b UPMT27 to develop a live attenuated FAdV vaccine via the gene-editing CRISPR/Cas9 technology. Primary specific pathogen-free (SPF) chicken embryonic liver cells (CELs) infected with the modified isolated (cfUPMT27) were reported with significantly reduced cytopathic effects, delayed viral localization into the nucleus, and low apoptotic rates. cfUPMT27 isolate also exhibited constant amino acid substitution of Y179D in subsequent passages. Meanwhile, the liver of cfUPMT27 inoculated-SPF chicken embryonic eggs (CEE) was observed with mild hydropericardium and reported with a delayed mortality at 6-days post-infection (dpi). This holistic, integrative study incorporating genetic, pathology, and immunology analysis proposed cfUPMT27 isolate as a candidate vaccine for FAdV infections, providing efficient future protection in chickens.

Hyaluronic acid (HA) is a key glycosaminoglycan in the extracellular matrix, essential for cellular signaling, hydration, and tissue homeostasis. This study identified two novel polysaccharide lyase family 33 (PL33) hyaluronate lyases, BxHly33 and BiHly33, from the human gut microbiome using metagenomic screening. These enzymes demonstrated high specificity and stability in degrading HA, with optimal activity at pH 6.6-7.6 and temperatures of 35-40 °C. Furthermore, structural and biochemical analyses revealed their catalytic mechanisms, highlighting key residues responsible for their function. Notably, specific alanine substitutions significantly enhanced their enzymatic activity. BxHly33 and BiHly33 present promising alternatives to conventional hyaluronidases, which are often costly and immunogenic, for drug delivery and tissue engineering applications. This study will provide novel insights into exploring their therapeutic potential in HA degradation therapies.

Emerging infectious diseases caused by various tick-borne microorganisms (TBMs) pose public and animal health concerns, including camels, with no defined global distribution. In this study, 150 blood samples and 288 ticks were collected from symptomatic two-humped camels (Camelus bactrianus) in Gaotai County, Gansu Province, China. Morphologically identified ticks were confirmed using cytochrome oxidase I (COI), and the findings revealed two species, Hyalomma asiaticum and Haemaphysalis longicornis (prevalence: 245/288 [88.19 %] and 34/288 [11.81 %], respectively). The extracted Genomic DNA from blood and ticks was processed by conventional PCR to investigate the existing TBMs based on 16S rRNA, 18S rRNA, and 17-kDa genes. Different TBMs, including Anaplasma bovis, Colpodella sp., Rickettsia rickettsii, and Candidatus Rickettsia jingxinensis, have been documented as single infections at different rates. High single infection rates (198/218; 90.83 % and 117/150; 78.00 %) of A. bovis in Hy. asiaticum and camel blood were recorded, whereas the lowest single infection rate (3/22; 13.64 %) of R. rickettsii was noted in Hae. longicornis. Co-infection with Rickettsia spp. + A. bovis (20/288; 6.94 %), Colpodella sp. + A. bovis (14/288; 4.86 %), Colpodella sp. + Rickettsia spp. (1/288; 0.35 %), and Colpodella sp. + Rickettsia spp. + A. bovis (1/288; 0.35 %) were recorded as concurrent infection. Phylogenetic analysis revealed that the representative TBMs have close similarities and clustered together with their corresponding isolates from China, South Korea, India, the USA, Mexico, Bangladesh, Malawi, Japan, Pakistan, Cyprus, Nigeria, Poland, and Brazil. These findings present a preliminary baseline regarding TBMs infection in camel blood and ticks and provide a framework for further studies on the prevalence and effective control measures for ticks and tick-associated diseases.

In the present study, the taxonomic position of Bacillus yapensis was evaluated using phylogenetic, amino acid identity (AAI) and average nucleotide identity (ANI) values. In the phylogenetic tree, Bacillus yapensis clustered with Robertmurraya species. The AAI value between Bacillus yapensis XXST-01T and Robertmurraya species was above the cut-off level (65%) for genus delineation. The ANI value between Bacillus yapensis XXST-01T and Robertmurraya species was below the threshold value (95-96%) for bacterial species delineation. Based on the results, we propose to transfer Bacillus yapensis to the genus Robertmurraya as Robertmurraya yapensis comb. nov. The type strain is XXST-01T (= MCCC 1A14143T = JCM 33181T).

This study reports the molecular identification of a tambacu-a hybrid of Colossoma macropomum and Piaractus mesopotamicus-in the floodplain of the upper Paraná River using molecular markers. The PCR-GEL method, based on the analysis of COI and TROP fragments, successfully characterized the hybrid, while PCR-SEQ, employing RAG2 and TROP markers, identified variable sites that differentiate the parental species and the hybrid.

Bacteria deploy a diverse arsenal of toxic effectors to antagonize competitors, profoundly influencing the composition of microbial communities. Previous studies have identified an interbacterial toxin predicted to exhibit proteolytic activity that is broadly distributed among gram-negative bacteria. However, the precise mechanism of intoxication remains unresolved. Here, we demonstrate that one such protease toxin from Escherichia coli, Cpe1, disrupts DNA replication and chromosome segregation by cleaving conserved sequences within the ATPase domain of type II DNA topoisomerases GyrB and ParE. This cleavage effectively inhibits topoisomerase-mediated relaxation of supercoiled DNA, resulting in impaired bacterial growth. Cpe1 belongs to the papain-like cysteine protease family and is associated with toxin delivery pathways, including the type VI secretion system and contact-dependent growth inhibition. The structure of Cpe1 in complex with its immunity protein reveals a neutralization mechanism involving competitive substrate binding rather than active site occlusion, distinguishing it from previously characterized effector-immunity pairs. Our findings unveil a unique mode of interbacterial intoxication and provide insights into how bacteria protect themselves from self-poisoning by protease toxins.

Acinetobacter baumannii stands out as a potent pathogenic microbe responsible for healthcare-associated infections characterized by elevated morbidity and mortality. This bacterium has acquired a range of mechanisms for resisting antibiotics, resulting in the emergence of strains that can withstand antibiotics from multiple classes. Effectively addressing this urgent concern requires finding ways to overcome these resistance mechanisms. In this context, our study focuses on TetR Transcriptional Factor Regulators (TetR-FTRs). It coordinates functions of tetracycline efflux pump proteins (TetA and TetR) and exert influence over metabolic pathways, quorum sensing, and biofilm formation. The primary objective is to identify potent inhibitors targeting TetR-FTRs through scaffold-based shape screening across thirteen distinct databases. A wide array of in silico techniques was employed, including molecular docking, molecular dynamics simulations, Swiss Similarity search, Virtual Screening, MM/GBSA analysis, ADMET assessment, PAINS assay, SIFT analysis, and MM/PBSA calculations. The initial Swiss similarity search yielded 2178 compounds for subsequent virtual screening, with the application of PAINS analysis rigorously pruning the list, eliminating 14 false positive hits. Further refinement through SIFT approach discriminated closely related interacting compounds into three distinct clusters - ChemBridge5963254, BDH33906706, and ZINC000013607604, which fulfilled all SIFT criteria. Comparative evaluation against reference compounds revealed favorable glide scores, lower binding free energies, and interactions with crucial active site residue Hsd128-Mg2+. Molecular dynamics simulations consistently exhibited stable binding for these clusters in contrast to reference compounds. Our analysis underscores three specific compounds, namely ChemBridge5963254, BDH33906706, and ZINC000013607604, as promising candidates for addressing tetracycline resistance and combating A. baumannii infections.

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b5-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root (LR) formation. Whereas the other Cytb5-HBD proteins in A. thaliana regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function and whether RLF orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme in vitro and that two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, we show that MpRLF, a RLF ortholog in the bryophyte Marchantia polymorpha, also binds to heme in vitro and that MpRLF rescues the LR formation phenotype of the A. thaliana rlf mutant. Mprlfge, the loss-of-function mutation in MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and reproductive organ formation. Our findings indicate that MpRLF is essential for proper vegetative and reproductive development in M. polymorpha. This suggests that RLF-dependent redox reaction systems are conserved across diverse plant species and were independently co-opted for organ development in bryophyte and seed plant evolution.

Bioinvasions represent a major environmental issue, particularly when they take place in biodiversity hotspots, such as mangrove ecosystems that serve as shelter for many marine species and fisheries resources. In this work, we used an integrative approach based on DNA and morphological analyses to identify individuals and the putative presence of cryptic diversity in the invasive tiger prawn (Penaeus monodon) along a mangrove area on the northern coast of Brazil, referred to as "Blue Amazon". A fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was selected for DNA Barcode and associated with distance-based (ABGD-Automatic Barcode Gap Discovery) and probabilistic (GMYC-Generalized Mixed Yule Coalescent and bPTP-Bayesian Poisson tree processes) species delimitation methods. Furthermore, the maternal origin of collected specimens was tracked. The molecular analyses recovered two genetically divergent lineages (7.7%) within the tiger prawn, indicating the occurrence of two species of this bioinvader on the northern coast of Brazil. Even though no differences in external morphology were detected, both lineages could be differentiated by their internal structures. The molecular traceability of the origin of samples showed that lineages I and II shared haplotypes with specimens from 11 and nine countries, respectively, including a shrimp breeding center in Vietnam. This is the first record of two species of tiger prawn along the Brazilian continental shelf. These findings are useful to the development of effective management policies in a region of particular biological relevance.

The taxonomic position of Microbacterium chocolatum was evaluated through 16S rRNA gene sequence and genome-based analyses. The pairwise similarity of the 16S rRNA gene sequence retrieved from the genome data between Microbacterium aurantiacum KACC 20510T and Microbacterium chocolatum JCM 12412T was 99.3%, while between Microbacterium kitamiense JCM 10270T and M. chocolatum JCM 12412T was 99.4%. The similarity between M. aurantiacum KACC 20510T and M. kitamiense JCM 10270T was 100%. In the phylogenomic tree (based on 71 bacterial single-copy genes), M. aurantiacum KACC 20510T, M. kitamiense JCM 10270T and M. chocolatum JCM 12412T showed no genetic divergence. The average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) values among M. aurantiacum KACC 20510T, M. chocolatum JCM 12412T and M. kitamiense JCM 10270T were above the cut-off values (95-96% for ANI and 70% for dDDH) for species delineation. This study supports the previous reclassification of Microbacterium kitamiense Matsuyama et al. 1999 as a later heterotypic synonym of Microbacterium aurantiacum Takeuchi and Hatano 1998, and also proposes that Microbacterium chocolatum Takeuchi and Hatano 1998 is a later heterotypic synonym of Microbacterium aurantiacum Takeuchi and Hatano 1998.

Aim. Antimicrobial resistance poses a critical global health threat, driven by the dissemination of resistance genes via mobile genetic elements (MGEs). This study aims to enhance the detection of MGE insertions in multidrug-resistant Escherichia coli by derivatizing the pBACpAK entrapment vector. Methods and results. Three derivatives were constructed with additional nucleotides upstream of the cI repressor gene, based on conserved regions identified from GenBank sequences containing known IS26 and IS1 insertions. Using colony PCR, intracellular transposition screening was performed on 194 tetracycline-resistant colonies from four E. coli ESI123 strains carrying different pBACpAK constructs. The derivatives showed increased MGE capture rates (10.7-73.1 %) compared to the WT vector (3.75%), identifying multiple MGEs, including the novel composite transposon Tn7824. Tn7824 harbours the bla OXA-181 carbapenem resistance gene and the qnrS1 quinolone resistance gene, highlighting the clinical relevance of these findings. Long-read sequencing of transposants confirmed the accuracy of MGE identification and structural characterization, which also revealed chromosomal integration events of the pBACpAK derivatives mediated by flanking insertion sequences. Conclusions. The modifications introduced in the pBACpAK derivatives could increase the detection of transposition events by alleviating spatial constraints, allowing for more robust MGE detection.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of morbidity and mortality worldwide in susceptible patients, particularly in those with respiratory disorders. The rising prevalence of multidrug-resistant strains and the failure of previous P. aeruginosa vaccine candidates in clinical trials highlight the urgent need to investigate novel vaccine antigens. In this study, we evaluated the protective potential of two antigen candidates, LptH and OprM, previously identified based on their involvement in host-cell attachment in a murine acute pneumonia model. Recombinant Escherichia coli BL21 clones overexpressing these proteins showed 8.8- and 3.5-fold increased attachment to 16HBE14o- cells in vitro, confirming their role in host-cell attachment. Immunisation with rLptH significantly reduced bacterial burden in the lungs by 1.12 log10 CFU and improved animal welfare scores compared to adjuvant-only controls. Serological and immunophenotyping analyses revealed that the monovalent rLptH vaccine stimulated antigen-specific IgG1 and IgG2c isotype production, and enhanced IFN-γ and IL-17 recall responses in the spleen. Moreover, a trivalent vaccine comprising rLptH and two other P. aeruginosa antigens, rFtsZ, and rOpmH, achieved a 2.33 log10 CFU reduction in lung bacterial burden, and 1.85 log10 CFU reduction in dissemination. These encouraging findings support the potential of LptH as a promising antigen for the development of a protective multivalent vaccine against P. aeruginosa infections.

Over the last decade, the emergence of aviadenovirus infections, namely inclusion body hepatitis (IBH) and gizzard erosion and ulceration syndrome (GEUS) have been occurring in Morocco with significant losses to the poultry industry. In this work, 255 suspected flock cases of IBH and GEUS were assessed among various poultry production types in the country from 2012 to 2024. Macroscopic pathological changes in affected chickens included an enlarged, discolored liver or a distended gizzard with haemorrhagic content and erosion/ulceration on the koilin layer. Microscopic findings revealed severe necrotic hepatitis, consistent with IBH, and/or necrotizing ventriculitis, which is characteristic of GEUS, with intranuclear viral inclusion bodies (INIB) in the hepatocytes or epithelial cells of the gizzard, which are attributable to fowl adenovirus (FAdV). The real time PCR results revealed that 71% (182/255) of the suspected FAdV cases tested positive. The prevalence varied by poultry production type, with broiler farms being the most affected at 79% (144/194), followed by layer farms at 15% (28/42), breeder farms at 5% (9/13), and turkey farms at 1% (1/6). Sequencing of the isolates identified FAdV serotype 11 (species D) and serotype 8b (species E) in 22 and 6 cases of IBH, respectively. Additionally, FAdV serotype 1 (species A) and serotypes 8a and 8b (species E) were detected in 9 and 2 cases of GEUS, respectively. These findings underscore the significant impact of fowl adenoviruses on the Moroccan poultry industry and highlight the need for continuous surveillance and targeted control interventions.RESEARCH HIGHLIGHTS Increase in FAdV infections (IBH and GEUS) in Morocco over past decade.FAdV-positive cases were mainly found in broiler farms (79%).Identification of FAdV-11 and 8b serotypes for IBH and FAdV-1, 8a and 8b for GEUS.

Bovine Kobuvirus (BKV) is an emerging pathogen associated with diarrhea in cattle. Limited reports on its prevalence and genetic characteristics are available. To determine the epidemiology and genetic evolution of BKV strains circulating in Yunnan Province, China, 204 diarrheal samples were collected from cattle farms across five regions for screening for BKV infection.

RT-PCR analysis identified 40 BKV-positive samples, yielding an infection rate of 19.6%. Positive samples were inoculated into Vero cells for continuous passage, followed by molecular biology, immunofluorescence, and electron microscopy identification. Two BKV strains, BKV YN-1 2023 and YN-2 2023, were isolated. Whole-genome sequencing revealed genome lengths of 8289 bp and 8291 bp (GenBank No. PV410179 and PV410180), respectively. Phylogenetic analysis demonstrated that both strains belong to genotype B, the dominant genotype circulating in China, and are closely related to the previously reported Chinese strain BKV 13/2021. The genetic similarity of two BKV isolates was analyzed. Genome-wide nucleotide identities ranged from 39.9 to 93.9%, with the highest similarity to BKV13 2021 CHN. ORF analysis showed nucleotide and amino acid similarities of 48.7-93.9% and 29.3-98.5%, respectively. Compared to the BKV13 2021 CHN, both isolates exhibited high conservation in VP0, VP3, and nonstructural proteins (97.8-100%), while the L protein had the lowest similarity (94.7-95.2%). The 5' UTR showed lower conservation than the 3' UTR, suggesting regulatory variations.

These findings reveal that the circulating BKV strains in Yunnan belong to the globally prevalent genotype B and are widely distributed. This study provides valuable insights into the molecular epidemiology, genetic diversity and evolutionary dynamics of BKV, offering an important reference for developing diagnostics, vaccines and further studies on its pathogenesis.

As emerging zoonoses represent a significant public health threat, understanding how pathogens' host ranges evolve is critical to protect human and wildlife health. Closely related hosts infected with host-specific pathogens provide valuable opportunities for clear inferences of host range evolution, as they allow for the examination of early diversification patterns in their resident pathogens. Pneumocystis, an obligate lung symbiont that is believed to be ubiquitous in mammals, exemplifies such a model. To explore the early stages of divergence in Pneumocystis, we collected geographically dispersed samples from two sister species of deer mice: Peromyscus leucopus (white-footed mice) and Peromyscus gossypinus (cotton mice). We sequenced two nuclear and two mitochondrial loci of Pneumocystis sampled from the lungs of these mice. These sequences revealed three distinct Pneumocystis taxa, two of which were found to cross-infect both host species and were often found coexisting within the same individual. Genetic diversity and phylogenetic analysis suggest that the three Pneumocystis taxa represent separate species. Further analysis of the mitochondrial large subunit rRNA gene from the most common taxon of these three revealed that host geographic origins influenced Pneumocystis genetic structure more than host species identity. Nevertheless, the results also suggest an overall interconnectedness of the symbiont metapopulation.

The study focused on the evaluation of the probiotic potency of Bacillus velenzensis STPB10, isolated from the digestive tract of the hillstream fish Snow trout (Schizothorax richardsonii). Ten B. velezensis strains were identified through colony morphology, biochemical analysis, and 16 S rRNA methods. The representative laboratory strain STPB10, was subjected to various in vitro assessments including tolerance to pH, bile salt resistance, NaCl, temperature, biofilm, endospore formation, antibiotic susceptibility and antagonistic activity to determine its potential as a probiotic strain. B. velezensis STPB10 exhibited positive results for catalase, glucose, arginine, lysine, and endospore tests, while it yielded negative results for oxidative/fermentative, DNase, and urease tests. This test isolate was γ-hemolytic in nature and demonstrated growth across a temperature range of 15 to 35 °C. Moreover, it exhibited survival rates of 31, 39.7, 47, 63, and 75% at pH values of 2, 2.5, 3, 3.5, and 4, respectively, after 6 h of incubation. At a bile salt concentration of 0.3%, the bacterium exhibited a survivability of more than 99%. A strong biofilm production by B. velezensis STPB10 was detected in tryptone soya broth enriched with 0.45% glucose (p < 0.05). It also exhibited significantly greater adhesion to intestinal mucus (63.67%). Following exposure of the intestinal mucus to B. velezensis STPB10, the adhesion of A. hydrophila, A. veronii, (A) salmonicida and V. anguillarum to the mucus was notably reduced. It was susceptible to several antibiotics and produced an antagonistic effect against pathogenic bacteria Aeromonas salmonicida, Aeromonas veronii, Vibrio anguillarum, and Aeromonas hydrophila isolated from diseased fish. The pathogenicity of (B) velezensis STPB10 through intraperitoneal injection and immersion challenge at cell concentrations of 108 and 109 CFU mL-1 revealed that the strain did not produce any pathogenic risk to common carp. These findings highlight the resilience and adaptability of B. velezensis STPB10 as a candidate probiotic in aquaculture.

The red-eared slider, Trachemys scripta elegans (Wied, 1938), has been introduced worldwide, partly because of the exotic pet trade in the 1980s and 1990s. When T. s. elegans is released or escapes into natural environments, it often establishes new feral populations due to its tolerance for a variety of aquatic ecosystems. Therefore, it is now considered one of the most invasive species in the world because it can compete with native turtle species. In the present study, our objectives were to identify the potential for polystome spillover and spillback resulting from the introduction of the red-eared slider into new environments in North America. Fieldwork investigations were thus conducted mainly in aquatic habitats in Florida and North Carolina, United States, but also in Connecticut, Indiana, Kansas, Maine, Nebraska and New York. Using DNA barcoding based on cytochrome c oxidase I (COI) sequences, we surveyed the species diversity of polystome within American freshwater turtles. These included T. s. elegans but also Apalone ferox, Apalone spinifera, Chelydra serpentina, Chrysemys picta, Kinosternon baurii, Pseudemys spp., Sternotherus minor and Sternotherus odoratus. Genetic evidence confirmed that invasive populations of T. s. elegans in southern Europe have transmitted their own polystomes to native host species following spillover effects, and revealed here that T. s. elegans in non-indigenous habitats in the United States acts as a new reservoir of infection for native polystomes following spillback effects, thus increasing indigenous parasite transmission in the wild. Together, these findings raise further concern about the spread of non-native turtles and their impact on parasite transmission.

Bacteria respond to osmotic stress by intracellularly accumulating low molecular weight compounds called compatible solutes, also known as osmolytes. Glycine betaine (N,N,N-trimethylglycine, GB) is a highly effective and widely available osmolyte used by bacteria, algae, and plants for abiotic stress protection. Here, we highlight the dual roles of GB, dimethyl glycine (DMG), and sarcosine for both osmoprotection and a less known role as sole carbon sources. First, we showed that the marine halophile Vibrio natriegens can grow in 1% to 7% NaCl and biosynthesize GB, ectoine, and glutamate and import GB, DMG, and sarcosine in response to osmotic stress. Betaine-carnitine-choline transporters (BCCTs) for the uptake of GB and DMG, but not sarcosine, were identified. Bioinformatics analyses uncovered homologs of GB, DMG, and sarcosine catabolism genes (dgcAB_fixAB, gbcA, gbcB, purU, soxBDAG, glyA, glxA) clustered in the V. natriegens genome, and these genes had a limited distribution among vibrios. We showed that V. natriegens ATCC 14048 grew on GB, DMG, and sarcosine as sole carbon sources, and gbcA and dgcA were required for growth. A contiguous catabolism cluster was present in a subset of Vibrio fluvialis strains, and we demonstrated the growth of V. fluvialis 2013V-1197 in DMG and sarcosine as sole carbon sources. Phylogenetic analysis revealed the catabolism cluster did not share a common ancestor among members of the family Vibrionaceae.IMPORTANCECompatible solutes are frequently the most concentrated organic components in marine organisms, allowing them to adapt to high saline environments as well as affording protection to other abiotic stresses. These organic compounds are significant energy stores that have been overlooked for their potential as abundant nutrient sources for bacteria. Our study characterized glycine betaine (GB), dimethyl glycine (DMG), and sarcosine catabolism genes and showed their efficient use as carbon and energy sources by marine halophilic vibrios.

The elongin BC complex (ELOBC) interacts with BC-box-containing proteins and plays a role in various cellular processes, including transcriptional regulation and ubiquitination. Elongin BC and polycomb repressive complex 2-associated protein (EPOP) contains a BC-box motif in its N-terminal region and influences cancer cell proliferation and differentiation. A previous study showed that a BC-box containing an EPOP-derived peptide suppresses cancer cell growth and induces apoptosis by disrupting the interaction between the ELOBC and its partner proteins. Here, we report the crystal structure of the EPOP BC-box peptide bound to the ELOBC and compare it with the structures of other BC-box-containing proteins in complex with the ELOBC. The overall structure of interactions between the BC-box and the ELOC was similar across different complexes, indicating a conserved binding mode. Our structural analysis revealed that the strictly conserved leucine residue (Leu40) within the BC-box of EPOP, which was previously suggested to be critical for interactions between the BC-box and the ELOC, was deeply embedded in the hydrophobic pocket of the ELOC protein. This study provided structural insights into BC-box-mediated protein-protein interactions and may serve as a fundamental resource for developing small molecules that modulate the interactions between ELOBC and BC-box-containing proteins.

This study aimed to characterize the duck circovirus circulating in Northern Vietnam based on complete genome sequences. Between 2023 and 2025, 45 pooled tissue samples were collected from nine duck flocks in several provinces in Northern Vietnam. Of the 45 samples tested, 16 (35.56%) were positive for the DuCV genome, as determined using conventional polymerase chain reaction. Nine representative strains were selected for viral genome sequencing. The results indicated that the complete Vietnamese DuCV genomes were from 1992 to 1995 bp in length, and the degree of nucleotide identity shared among them ranged from 96.88% to 99.84%. Phylogenetic analysis of the complete genomes showed that the nine Vietnamese DuCV strains belonged to genotype I, subgenotypes Ia (two strains), Ib (four strains), and Ic (three strains). These viral strains were genetically related to viruses reported in China from 2019 to 2023. Recombination events occurred on the Cap gene sequences of three Vietnamese DuCV strains (Vietnam/VNUA-102/2023, Vietnam/VNUA-225/2023, and Vietnam/VNUA-318/2024). One positive selection was detected on the Rep protein sequence.

Bacterial chemoreceptors sense extracellular stimuli and drive bacteria toward a beneficial environment or away from harm. Their ligand-binding domains (LBDs) are highly diverse in terms of sequence and structure, and their ligands cover various chemical molecules that could serve as nitrogen, carbon, and energy sources. The mechanism of how this diverse range of LBDs senses different ligands is essential to signal transduction. Previously, we reported that the chemoreceptor MCP2201 from Comamonas testosteroni CNB-1 sensed citrate and L-malate, altered the ligand-free monomer-dimer equilibrium of LBD to citrate-bound monomer (with limited monomer) and L-malate-bound dimer, and triggered positive and negative chemotactic responses. Here, we present our findings, showing that D-malate binds to MCP2201, induces LBD dimerization, and triggers the chemorepellent response exactly as L-malate did. A single site mutation, T105A, can alter the D-malate-bound LBD dimer into a monomer-dimer equilibrium and switch the negative chemotactic response to D-malate to a positive one. Differences in attractant-bound LBD oligomerization, such as citrate-bound wildtype LBD monomer and D-malate-bound T105A dimer, indicated that LBD oligomerization is a consequence of signal transduction instead of a trigger. Our study expands our knowledge of chemoreceptor-sensing ligands and provides insight into the evolution of bacterial chemoreceptors.

The species of the genus Aggregata are intracellular parasites that infect cephalopods. In this study, A. valdessensis descripton is expanded from cecum and intestine samples of Octopus tehuelchus by in vivo observations as well as scanning and transmission electron microscopy of gametogonic and sporogonic stages. Infection in O. tehuelchus is noticeable as white spherical cysts, visible to the naked eye. Additionally, merogony stages were found in field-collected Cyrtograpsus spp. crabs, and described from histological slides and TEM images. We established two transmission paths by coproparasitological analyses and experimental infections, confirming that the crustaceans acquire infection by feeding on octopus feces, but also by scavenging octopuses' dead bodies. Genetic analyses using the 18S molecular marker from octopus and crab stages allowed us to to confirm that they are conspecific, establishing the complete life cycle of A. valdessensis. For the first time in the southern hemisphere, a life cycle for the genus Aggregata is elucidated, being the second worldwide.

Life's organic molecules are built with diverse functional groups that enable various importance biological functions. As such, the discovery of unique functional groups in nature can expand our understanding of the natural world. Here we report the genome-aided discovery of sulfenicin, a polyketide-non-ribosomal peptide hybrid natural product from a marine Streptomyces bacterium bearing a unique acylsulfenic acid functionality. Through a series of heterologous biosynthesis, functional genetics and enzymatic reconstitution experiments, we show that this previously described synthetic functional group is biologically assembled by a set of enzymes from both primary and secondary metabolism, including a flavin-dependent S-hydroxylase that hydroxylates the sulfur atom of a thiocarboxylic acid. Although public databases so far include no parallel for the sulfenicin biosynthetic gene cluster, enzymes catalysing the production of acylsulfenic acid are widely distributed in bacterial genomes, implying that this labile functional group may similarly have a broad distribution among specialized metabolites.

This study investigates mastitis in the dairy industry, with a focus on the issue of antibiotic resistance. This study was designed to evaluate mastitis prevalence and investigate the bacteriological profiles of subclinical mastitis (SCM) milk, mastitis-free milk, and market milk. Out of 374 quarter milk samples, 26.2 % were from animals with SCM. Bacteriological examination identified 87 Gram-negative bacterial strains from subclinical mastitis milk (SCMM) (42.9 %), subclinical mastitis-free milk (SCMFM) (17.97 %), and market milk (MM) (58 %). MALDI-TOF MS identified species including E. coli, K. pneumoniae, Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, and Acinetobacter baumannii, with E. coli being the most frequent. Multi-drug resistant (MDR) phenotype was found in 43.7 % of isolates, with 57.1 % from SCMM, 43.8 % from SCMFM, and 24.1 % from MM. Biofilm production was observed in 44.8 % of isolates, with a significant correlation between MDR and biofilm formation. Eight strains (9.2 %) were extended-spectrum β-lactamases (ESBLs) producers, with blaCTX-M, blaTEM, and blaSHV genes detected. A. baumannii harbored multiple resistance genes, including blaTEM, blaCTX-M, blaOXA51, blaOXA23, and blaNDM, showing both phenotypic and genotypic ESBLs and carbapenemase activity. The presence of MDR, ESBLs, and carbapenemase producing Gram-negative bacteria in SCMM, SCMFM, and MM indicates a concerning exchange of bacteria and antimicrobial resistance genes between human and animal hosts, posing risks of milk contamination and environmental hazards. A one-health approach is essential for controlling antimicrobial-resistant bacteria, emphasizing prudent antimicrobial use in human and animal healthcare, and improving farm hygiene practices.

Lacticaseibacillus species have shown potential in managing hyperglycemia, hypercholesterolemia, and oxidative stress, depending on the strain and species. This study aimed to isolate a novel Lacticaseibacillus rhamnosus strain from healthy newborns and assess its hypoglycemic and antioxidative activity, along with other probiotic properties. A non-hemolytic L. rhamnosus LBUX2302 was isolated, and it exhibited survival rates of 2.7%, 22%, and 27.5% at pH 2, 3, and 5 for 120 min. It metabolized various carbon sources and showed resistance to gentamicin, dicloxacillin, and penicillin; coaggregated with Salmonella typhi ATCC14028, Staphylococcus aureus STCC6538, and Escherichia coli O157:H7. L. rhamnosus LBUX2302 showed hydrophobicity, autoaggregation, and adhesion to HaCat, HeLa, MCF-7, SK-LU-1, and SW620 cell lines. It also exhibited extracellular activity of bile salt hydrolase. Enzymatic inhibition assays revealed 66% and 24% inhibitions of α-amylase and α-glucosidase, respectively. Its cell-free supernatant inhibited DPPH (89%), hydroxyl (81%), and superoxide anion radicals (61%). Also, antioxidant activity was observed in whole cells and cell fragments. Finally, the presence of ferulic acid activity was detected. The results highlight L. rhamnosus LBUX2302 as a promising probiotic with hypoglycemic and antioxidant effects, warranting further in vivo evaluation for its possible inclusion in functional food and health formulations.

Black flies (family Simuliidae) are globally distributed insects of great medical and veterinary importance. However, little is known about their mitogenomics. Therefore, in this study, the mitochondrial genomes (mitogenomes) of five black fly species‒Simulium bidentatum, S. siripoomense, S. fenestratum, S. chamlongi, S. quinquestriatum‒were sequenced using next-generation sequencing. We conducted a comprehensive comparative analysis of these mitogenomes focusing on sequence length, A + T content, A/T bias, A + T- rich regions, overlapping and intergenic regions, nucleotide composition, relative synonymous codon usage, and the non-synonymous/synonymous substitution ratio (Ka/Ks). Additionally, we analyzed the phylogenetic implications in combination with the Simuliidae and Nematocera species available in GenBank. The mitogenomes ranged from 15,739 to 16,451 base pairs (bp); each contained 37 genes, with no gene rearrangements. The tRNASer (Ser1) lacks the dihydrouridine arm. Selection pressure analysis of 13 PCGs in 45 Nematocera mitogenomes revealed that nd2, nd5, nd4 l, and nd1 had Ka/Ks ratios greater than 1, indicating higher rates of non-synonymous substitutions. In contrast, cox1 showed the lowest Ka/Ks values, indicating strong purifying selection. Phylogenetic analyses supported the monophyly of each subgenus within Simulium, but revealed different subgeneric relationships compared to previous studies. We also observed that different samples can yield different phylogenetic results for higher level relationships within Culicomorpha. The phylogenetic position of Anisopodidae within the Bibionomorpha remains unclear and warrants further investigation.