Tissue ontogeny and chemical composition influence bacterial biodiversity in the wood and shoot tip of Populus nigra.

Plant-microbe interactions significantly influence plant growth dynamics and adaptability. This study explores the impact of metabolites on microbial biodiversity in shoot tips and wood of Populus nigra under greenhouse conditions, using high-throughput sequencing and metabolite profiling. Branches from P. nigra were harvested, rooted, and transplanted into pots for growth. After 3 months, tissue samples from shoot tips and wood were collected, and metabolites extracted and analysed using GC-MS and LC-MS. Genomic DNA was extracted and subjected to high-throughput sequencing for bacterial biodiversity profiling. Both datasets were analysed using bioinformatic and statistical pipelines. Metabolite profiling indicated that shoot tips had a higher relative abundance of primary and secondary metabolites, including sugars, fatty acids, organic acids, phenolic acid derivatives and salicinoids, while wood was enriched in flavonoids. Bacterial biodiversity also differed significantly between these tissues, with Clostridiales, Bacteroidales and Bacillales dominating in shoot tips, associated with rapid growth and anaerobic fermentation, while wood tissues were characterized by diazotrophs from Rhizobiales, Sphingomonadales and Frankiales. PCoA clustering confirmed tissue-specific microbial differences. Functional analysis revealed an enrichment of fundamental cellular processes in shoot tips, while wood exhibited pathways related to degradation and mortality. Metabolite profiling revealed significant variations in primary and secondary metabolites, highlighting their influence on microbial biodiversity across plant tissues. The dominance of specific bacterial orders and distinct functional pathways in each tissue suggests a tailored microbial response to the unique environments of shoot tips and wood.

Barcoding of Italian mosquitoes (BITMO): generation and validation of DNA barcoding reference libraries for native and alien species of Culicidae.

BACKGROUND: Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). METHODS: A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. RESULTS: Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. CONCLUSIONS: This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.

REDESCRIPTION OF HASSALSTRONGYLUS ADUNCUS CHANDLER, 1932 (NEMATODA: HELIGMONELLIDAE), WITH THE DESCRIPTION OF A NEW SPECIES OF THE GENUS AND THEIR PHYLOGENETIC POSITION WITH OTHER HELIGMOSOMOIDEA TRAVASSOS, 1914.

ABSTRACT: Hassalstrongylus Durette-Desset, 1971 (Nematoda: Heligmonellidae), includes 19 species that are distributed from the southwestern United States to central-western Argentina. Hassalstrongylus aduncus is a parasitic nematode of rodents from the subfamilies Arvicolinae, Murinae, and Sigmodontinae, and has been recorded from southern Virginia and Oklahoma to Costa Rica. This species was described by Chandler in 1932; the morphology of the synlophe was not included. Subsequently, in 1972, Durette-Desset described only the synlophe of the middle region of the body in both sexes. Despite its wide geographical distribution, to date, there has been no redescription that includes information complementary to the morphology of the synlophe, such as a study of the body surface or a molecular phylogenetic analysis. We reevaluated the morphology of some specimens that were presumably similar to H. aduncus parasite of Sigmodon sp. from Jalisco, Mexico, and it was determined that these corresponded to an undescribed species of the genus. Herein, we present a redescription of H. aduncus parasite of Sigmodon toltecus from Hidalgo, Mexico, with morphological traits such as the excretory pore, deirids, and ovijector, and provide a description of the synlophe in the anterior and posterior regions of both sexes and include scanning electron microscopy images. Hassalstrongylus geolayarum n. sp. is differentiated from H. aduncus by the number of ridges in the middle region of the body (23 vs. 21), as well as proportions between some traits of males and females such as total length/spicule length, total length/gubernaculum length, total length/length of the esophagus and total length/distance of the vulva and the size of the eggs (42 vs. 58 µm). Phylogenetic analysis is based on partial sequences of the nuclear ribosomal internal transcribed spacer region (ITS1 + 5.8S + ITS2) of the rDNA, using the maximum-parsimony, maximum-likelihood, and Bayesian inference methods revealed the close relationship of H. aduncus + H. geolayarum n. sp. within the Heligmosomoidea and confirmed the placement of the Hassalstrongylus monophyletic clade well-supported within Heligmonellidae. The new species presented a genetic divergence of 3.4-3.8% relative to H. aduncus. This is the first species of the genus described in Mexico. Presumably, there are more species not yet described throughout the geographic range of H. aduncus. A taxonomic review and molecular phylogenetic analysis are required in which more species and genes are analyzed in Heligmosomoidea to confirm the status of the nonmonophyletic groups recovered here.

Production, Characterization, Kinetics, and Thermodynamics Analysis of Amyloglucosidase from Fungal Consortium.

The current study aimed to produce an amyloglucosidase enzyme from the fungal consortium. The best amylolytic fungal consortia were identified as Alternaria alternata and Aspergillus niger through the 18S rDNA technique. Fermentation kinetics and various nutritional and cultural parameters were analyzed. Maximum production was obtained in M4 media, pH 5.5, 30 °C, and 4 mL inoculum at 150 rpm after 72 h of incubation. Along with that, sodium nitrate at 2.5%, maltose, beef extract 1%, zinc sulfate (0.1%), and Tween 80 (0.1%) supported the maximum amyloglucosidase production. Amyloglucosidase was partially purified up to 1.6 purification fold with a specific activity of 1.84 Umg-1 in a stepwise manner by ammonium sulfate purification, dialysis, and ion exchange chromatography. The AMG enzyme also revealed maximum activity at 50 °C with 5.0 pH. Upon the kinetic analysis, the specific yield coefficient Yp/x and volumetric rates Qp and Qx were also found to be significant in the above optimized conditions. The Km value 0.33 mg mL-1 and Vmax 26.31 U mL-1 were obtained at 1% soluble starch substrate. Thermodynamic parameters for soluble starch hydrolysis were as follows: ΔH = 48.78 kJ mol-1, (Ea) = - 46.0 kJ mol-1, and ΔS = - 43.10 J mol-1 K-1. This finding indicates the indigenously isolated fungal consortium can be the best candidate for industrial applications.

Characterization of Intestinal Flora in Osteoporosis Patients Based on 16S rDNA Sequencing.

Aim: This study investigated differences in gut flora between osteoporosis (OP) patients and healthy individuals using 16S rDNA sequencing. The correlation between differential flora abundance and bone mineral density (BMD) was analyzed, and key flora and potential mechanisms associated with OP were explored. Methods: Forty-three OP patients and twenty-four healthy volunteers were recruited. Gender, age, height, weight, and BMD data were collected. DNA from fecal samples was extracted for 16S rDNA sequencing. The Kruskal-Wallis test assessed differences in gut flora composition, while LEfSe analysis identified significant flora. Spearman correlation analysis examined the relationship between differential flora and BMD, and PICRUSt predicted pathways involved in OP. Results: Significant differences in microbial composition were found between the two groups. Klebsiella, Escherichia-Shigella, and Akkermansia were biomarkers in OP patients, with Faecalibacterium in the healthy group. Akkermansia abundance negatively correlated with lumbar BMD, while Klebsiella and Escherichia-Shigella negatively correlated with femoral neck and hip BMD. Faecalibacterium showed a positive correlation with BMD. Functional predictions indicated differences in metabolism-related pathways between the groups. Conclusion: Gut flora differed significantly between OP patients and healthy individuals. Akkermansia, Klebsiella, and Escherichia-Shigella could serve as diagnostic biomarkers for OP, highlighting the potential of gut flora in OP diagnosis and treatment.

Protein UFMylation regulates early events during ribosomal DNA-damage response.

The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ataxia-telangiectasia-mutated (ATM)-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA-damage response in human cells. We further show the only ubiquitin-fold modifier 1 (UFM1)-E3 ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. Moreover, analysis of nuclear and nucleolar UFMylation targets in response to DSB induction further identifies key DNA-repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provide evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

Molecular phylogenetic analysis and seasonal dynamics of Eimeria species infecting broilers of Kashmir, India.

Globally, the poultry industry is seriously threatened by coccidiosis caused by various species of Eimeria. This protozoan parasite inhabits the epithelial lining of the gastrointestinal tract of poultry globally and can cause serious clinical disease. The present study was carried out on poultry farms located in various regions of Kashmir, India, to investigate the prevalence and phylogenetic relationships of Eimeria species affecting broiler chickens. Over a period of one year, fecal samples were collected from 60 poultry farms in Kashmir and morphological and molecular techniques were employed for Eimeria species identification. Results revealed a high prevalence of coccidiosis, with 58.3% (35/60) of farms positive for Eimeria. The most prevalent species were E. tenella (31/35, 88.6%) followed by E. acervulina (25/35, 71.4%), E. maxima (19/35, 54.3%), E. mitis (18/35, 51.4%), and E. necatrix (9/35, 25.7%). Seasonal variation in prevalence was also observed, with the highest rates in autumn (86.7%) and summer (66.7%). Additionally, younger birds (3-4 weeks) exhibited higher infection rates (85.7%) compared to older birds (57.9%) (5-6 weeks). Mixed infection was found in 94.2% (33/35) of positive farms. Phylogenetic analysis using ITS1 sequences confirmed species clustering and revealed evolutionary relationships among Eimeria species. E. tenella and E. necatrix formed a distinct clade, while E. acervulina formed another. The study underscores the importance of molecular techniques in accurate species identification and provides valuable insights into the epidemiology of coccidiosis in poultry in Kashmir. Effective control strategies, including vaccination and improved management practices, are necessary to mitigate the economic losses associated with this widespread poultry disease.

Revision of the Most Primitive Taxa of the Family Gyrodactylidae (van Beneden et Hesse, 1864) (Platyhelminthes, Monopisthocotyla) Based on ITS rDNA Phylogeny.

BACKGROUND: For the past 25 years, the ITS rDNA (ITS1-5.8S-ITS2) of Gyrodactylidae has been crucial for species identification, description, and phylogeny. This family includes 25 genera parasitizing marine and freshwater fish, initially distinguished by morphological differences in attachment and/or male copulatory organs. Gyrodactylus Nordmann, 1832, the most species-rich genus, has approximately 500 described species and an additional 25,000 species suspected. The genus is not monophyletic, and the functionally adaptive nature of morphological diagnostic characters complicates the delimitation of new genera. METHODS: A phylogeny based on ITS rDNA was proposed to address these challenges, using only complete sequences of primitive taxa. Fifty-four sequences were aligned with the MUSCLE v5.1 algorithm, creating a 1590 ps long matrix. Maximum Likelihood (ML) and Bayesian Inference (BI) methods with the models TVM+F+G4 and SYM+G4 for ITS1-ITS2 and 5.8S, respectively, were inferred using IQ-TREE v2.3.5 and BEAST v2.7.6.0. RESULTS: The findings revealed eleven main lineages. Four of them are proposed for classification into new genera: Cichlidarus gen. nov., Iraqemembranatus gen. nov., Macracanthus gen. nov., and Rysavyius gen. nov. Elevating the subgenus G. (Gyrodactylus) to genus rank was supported. CONCLUSIONS: The presented phylogeny provides a foundation for developing a classification system within Gyrodactylidae that is both reasonable and comprehensive.

Discovery of adults of the gorgoderid trematode Cercaria duplicata with first morphological description, molecular identification and notes on host specificity.

Rhopalocercous Cercaria duplicata von Baer, 1827 develops in an intermediate host, the unionid bivalve Anodonta anatina (L.), but its adult form has been unknown. We examined eight fish species occurring in the presence of a highly infested population of A. anatina in the Zeslawice reservoir (S Poland). Gravid Phyllodistomum specimens were obtained from the ureters of ide, Leuciscus idus (L.) and common rudd, Scardinius erythrophthalmus (L.). One of the rudd specimens was doubly infected, a trematode was also found in the urinary bladder. In addition, a gravid Phyllodistomum specimen was found in the ureter of a tench Tinca tinca (L.), caught in Lake Ilmedas (Lithuania). In order to clarify the phylogenetic position of larval and adult gorgoderids and to establish their life cycle, ITS2 and 28S rDNA sequences were analysed. The analysis showed that adult Phyllodistomum specimens located in the ureters are conspecific with C. duplicata. The trematode found in the urinary bladder of S. erythrophthalmus was P. folium (Olfers, 1816). It is suggested that adult stages of C. duplicata should be referred to as Phyllodistomum duplicatum n. comb. The intercaecal position of the uterus and the deeply-lobed ovary are the main features distinguishing it from other Phyllodistomum species. Host specificity and ecology are discussed.

The role of DNA topoisomerase 1α (AtTOP1α) in regulating arabidopsis meiotic recombination and chromosome segregation.

Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( A t T O P 1 α ) in male reproductive development of Arabidopsis thaliana, we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of A t T O P 1 α to meiosis. Our results demonstrate that the absence of A t T O P 1 α leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that A t T O P 1 α plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells.