Search Results (173)

Helminth infections remain a major constraint to livestock productivity, particularly in regions where domestic animals and wildlife share grazing habitats. This study investigated the molecular diversity and transmission dynamics of helminth communities in sheep (Ovis aries) and saiga antelope (Saiga tatarica) in West Kazakhstan. A total of 35 animals (20 sheep and 15 saiga) were examined, and helminths were identified using polymerase chain reaction targeting the ITS1 region of ribosomal DNA for nematodes and the mitochondrial cox1 gene for cestodes. Of the 20 analyzed samples, 80% were successfully identified at the molecular level. Detected species included Haemonchus contortus, Trichuris ovis, Chabertia ovina, Moniezia expansa, and Avitellina centripunctata. Phylogenetic analysis revealed that Chabertia ovina isolates from both hosts clustered within a single monophyletic clade, indicating high genetic similarity and supporting potential cross-species transmission. Mitochondrial markers provided higher resolution for cestode differentiation, whereas ITS1 was effective for nematode identification. The predominance of Chabertia ovina in saiga suggests ecological adaptation and efficient transmission within wild populations. These findings highlight the epidemiological significance of shared grazing ecosystems and underscore the need for integrated parasite control strategies that consider both livestock and wildlife reservoirs. Full article

Environmental DNA (eDNA) metabarcoding has emerged as a powerful tool for biodiversity monitoring, yet its accuracy is fundamentally constrained by the completeness and taxonomic reliability of reference sequence databases. For the Three Gorges Reservoir (TGR), no integrated multi-marker eDNA reference library exists, hampering standardized fish conservation monitoring under the Yangtze River Ten-Year Fishing Ban. Here, we constructed a comprehensive, multi-marker eDNA reference database for the fish fauna of the TGR, encompassing mitochondrial 12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I (COI) gene sequences from 173 specimens (120 species) collected between 2021 and 2024. After integrating publicly available sequences, the final database comprised 161 species. Then, we quantitatively compared species annotation performance between this local database and public repositories. Results showed that while public databases achieved higher nominal species coverage (94.67%), they exhibited critical deficiencies in annotation accuracy, correctly annotating only 77.97% (12S rRNA), 75.00% (16S rRNA), and 38.14% (COI) of sequences from shared species under controlled conditions. In contrast, the local database exhibited 92.37%, 93.10% and 100% annotation accuracy for the respective markers. Optimal interspecific Kimura 2-parameter (K2P) thresholds for species delimitation were 0.00448 (12S rRNA), 0.00531 (16S rRNA), and 0.00734 (COI). In addition, 15, 0, and 4 species pairs exhibited zero interspecific distance for 12S rRNA, 16S rRNA, and COI, respectively. These limitations reinforce the need for cautious interpretation of eDNA metabarcoding results and the integration of multiple markers or complementary nuclear loci. This study provides preliminary evidence that regionally curated, multi-marker reference libraries could improve taxonomic assignment reliability in eDNA metabarcoding compared to uncurated public repositories, providing a foundational resource for biodiversity conservation. Full article

Background: Pangolins are critically endangered mammals, and a comprehensive understanding of their genetic diversity is crucial for effective conservation. The mitochondrial genome serves as a vital molecular marker for phylogenetic and population genetic studies. Obtaining genetic material from these elusive animals non-invasively remains a challenge. This study aimed to sequence and characterize the complete mitochondrial genome of Manis javanica and explore the phylogenetic relationships among pangolin species. Methods: The complete mitochondrial genome was sequenced from a saliva-derived sample. Standard procedures for DNA extraction, amplification, and sequencing were employed. The genome was assembled and annotated using bioinformatic tools. Phylogenetic analysis was conducted based on the cytochrome c oxidase subunit I (COXI) gene sequences from nine pangolin species, with the resulting tree constructed using the maximum-likelihood method. Results: The complete mitochondrial genome of M. javanica (GenBank accession: PP110760) is a circular molecule of 16,573 bp, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The overall base composition showed a lower GC content (43.83%) than AT content (56.17%). Phylogenetic analysis based on COXI sequences delineated the nine species into three distinct genera: Manis, Phataginus, and Smutsia. Within the genus Manis, Manis pentadactyla was identified as the closest relative to M. javanica. The newly described species Manis mysteria was found to be closer to Manis culionensis and Manis crassicaudata than to other congeners. Furthermore, the analysis indicated that African pangolins diverged earlier than Asian pangolins. Conclusions: This study successfully demonstrates the feasibility of extracting and sequencing the complete mitochondrial genome from saliva samples, providing a valuable non-invasive method for future genetic studies on pangolins. The genomic data and phylogenetic results offer significant molecular insights that will benefit the genetic management and conservation of critically endangered pangolin resources. Full article

Populations of several billfish species are declining due to overfishing and bycatch, and fundamental aspects of their biology and population dynamics remain poorly understood. We provide the first assessment of the population genetic structure of longbill spearfish (Tetrapturus pfluegeri) in the western Atlantic Ocean. We screened variation at 12 nuclear microsatellite loci (n = 144) and mitochondrial DNA control region sequences (mtCR, n = 177). Both marker types revealed three genetically differentiated clusters, with mean values for microsatellites showing differentiation of F_ST = 0.136 and D_EST = 0.201, and for mtCR F_ST = 0.645. Microsatellite markers demonstrated moderate-to-high genetic diversity, with a mean allelic richness of 6.73 alleles per locus, moderate heterozygosities (Ho = 0.446, He = 0.604), and a positive inbreeding coefficient (F_IS = 0.22) across the three sample collection sites. The overall estimated effective population size was 789.2 (95% CI: 246.7–∞). The mtCR exhibited 96 haplotypes, with high haplotype (0.989 ± 0.003) and nucleotide (0.025 ± 1.3%) diversities. We found higher mean relatedness within clusters than among them, supporting the interpretation of population subdivision and the Wahlund effect. Tajima’s D and Fu’s Fs were negative across all localities, with significant values observed along the Brazilian coast but not in the Caribbean Sea. These neutrality test results, together with low Harpending’s raggedness indices from DNA sequence mismatch distributions, are consistent with historical demographic expansion. Our findings establish a genetic baseline for fishery monitoring and management, contributing to the conservation of T. pfluegeri populations in the western Atlantic Ocean. Full article

Background: Wolfram syndrome (WFS) is a rare neurodegenerative disease that is genetically determined and inherited in an autosomal recessive manner. Although the first clinical symptom appearing in early childhood is diabetes mellitus, subsequent symptoms are associated with optic nerve atrophy, followed by central nervous system atrophy. Methods: The aim of the study was to analyse magnetic resonance images (MRI) of the brain in combination with single-voxel magnetic resonance spectroscopy (MRS) and to assess the copy number of mitochondrial DNA (mtDNA-CN) in 10 patients with WFS compared with a control group of 17 healthy individuals. Results: A significant decrease in the amount of selected metabolites was observed in WFS patients compared to controls in all assessed brain regions (pons, cerebellum, white matter, thalamus, and hippocampus). For three metabolites, Glutamate (Glu), Glutamate + Glutamine (Glx) and total N-acetylaspartate (TNAA), significant differences in concentrations were found between the study groups in almost all matrices evaluating specific areas of the brain (p < 0.011), with the exception of a trend toward reduced TNAA in the hippocampus (p = 0.065). In addition, patients with WFS had a significant decrease in the mitochondrial-to-nuclear DNA ratio compared to controls (p < 0.0003). Some metabolites, such as N-acetylaspartate and total N-acetylaspartate, showed strong correlations with specific regions of the visual pathway on MRI scans in patients with WFS. Conclusions: Selected brain metabolites and mtDNA-CN may become potential markers of WFS, and the results of this study may be used to define indicators for future therapeutic strategies. Full article

Postglacial expansion dynamics strongly influence the genetic structure of temperate species; however, mitochondrial data from the Baltic region are limited. To assess diversity, phylogenetic origins, and regional structuring, we analyzed mitochondrial cytochrome b (726 bp) and control region (421–422 bp) sequences of the common vole (Microtus arvalis Pallas, 1779) from Lithuania. Of the 91 cytb sequences and 70 control region sequences analyzed, five and four haplotypes were identified, respectively. Markedly low haplotype and nucleotide diversity compared with most European populations were detected. Phylogenetic Maximum Likelihood and network analyses revealed that all Lithuanian haplotypes belong to the eastern European lineage and are most closely related to Polish and central European samples, which supports recolonization from a Carpathian refugium. Despite the overall low variation, we detected two distinct mitochondrial groups: a highly differentiated western group and a second group encompassing eastern, northern, and central–southern populations. This strong regional structuring suggests limited maternal gene flow on a small geographic scale. There was no evidence of introgression from related taxa, such as Microtus obscurus. Our findings refine the phylogeographic context of Baltic M. arvalis and highlight the region’s role in shaping postglacial diversity patterns. Full article

Background/Objectives: Although the etiopathogenesis of autism spectrum disorder (ASD) remains incompletely elucidated, current evidence supports a multifactorial model involving genetic and environmental factors that interact to induce a heterogeneous range of symptoms. In recent years, epigenetic mechanisms, particularly DNA methylation, have been recognized as key contributors to ASD pathophysiology. Alterations in mitochondrial DNA (mtDNA) methylation are also emerging as relevant contributors in several human conditions. The mitochondrial D-loop, a non-coding control region essential for mtDNA replication and transcription, is considered a hotspot for epigenetic regulation and its methylation levels have been found altered in various diseases, such as cancer, metabolic disorders, and neurological illness. However, to date, no studies have investigated mtDNA methylation changes in ASD. Methods: We analyzed the average methylation levels of a fragment containing ten CpG sites within the D-loop region and the mtDNA copy number in peripheral blood samples from 49 children with ASD and 50 neurotypically developing (NT) controls using Methylation-Sensitive High-Resolution Melting and quantitative PCR. Results: No significant differences in D-loop methylation levels were observed between ASD and NT children. Similarly, the mtDNA copy number did not differ between the two groups. No significant correlations were found between D-loop methylation or mtDNA copy number and either ASD severity or age. Conclusions: This is the first study investigating mtDNA methylation in ASD. Our results indicate that methylation of the D-loop region and the mtDNA copy number are not altered in ASD children. Further studies including larger cohorts and extended mtDNA regions are warranted to confirm and expand these findings. Full article

Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article

The blackfin flounder (Glyptocephalus stelleri) is an important demersal fish species widely distributed in the Northwest Pacific and represents a valuable fisheries resource in Korea and Japan. Understanding the genetic diversity and population connectivity of exploited marine species is essential for effective fisheries management and conservation. In this study, mitochondrial DNA control region sequences (401 bp) were analyzed from 62 individuals collected from Jumunjin, Korea, and Maizuru, Japan, to assess genetic diversity, demographic history, and population structure. Sequence analysis identified 48 haplotypes, revealing exceptionally high haplotype diversity (h = 0.982 ± 0.010) and relatively low nucleotide diversity (π = 0.011 ± 0.006). Neutrality tests and mismatch distribution analyses indicated a historical demographic expansion during the Pleistocene. No significant genetic differentiation was detected between the two sampling locations (F_ST = −0.004, p > 0.05), suggesting strong genetic connectivity between Korean and Japanese samples based on mitochondrial DNA data. These findings provide important baseline genetic information for understanding population connectivity and may contribute to the coordinated management of blackfin flounder fisheries in the Northwest Pacific. Full article

Background/Objectives: Mitochondrial DNA (mtDNA) analysis is an essential tool for human identification in contexts such as disaster victim identification (DVI) and missing persons cases, where the remains may be highly degraded or even skeletonised. Traditionally, capillary electrophoresis (CE)-based Sanger sequencing has been the standard method for analysing the mtDNA control region. With the development of massively parallel sequencing (MPS) technologies, mtDNA sequencing using MPS offers advantages over traditional Sanger sequencing, such as increased sensitivity, higher throughput, and less sample consumption. The Ion Chef™ and Ion S5™ XL system from Thermo Fisher Scientific represents one such MPS system. Methods: We conducted an internal validation study evaluating key parameters including (a) concordance, repeatability and reproducibility; (b) potential cross-contamination; (c) sensitivity; (d) effects of library pooling on read depth; and (e) mixture sample analysis. Additionally, to mimic samples typically encountered during forensic investigations, case type samples were also used to evaluate the performance of this workflow. While the entire mitochondrial genome was sequenced in this validation study, considering that the international guidelines for full mtDNA genome analysis and interpretation have yet to be fully updated, our analysis, interpretation and subsequent implementation are limited to the control region only. Results: The results obtained demonstrated the reliability, sensitivity and reproducibility of this MPS workflow. Conclusions: This internal validation study supports the implementation of this workflow in our laboratory for the analysis of forensic casework samples. Full article

Pancreatic cancer (PC) is a highly aggressive malignancy characterized by limited opportunities for early diagnosis and poor clinical outcomes, underscoring the need for minimally invasive biomarkers to improve detection and patient stratification. Given emerging evidence that mitochondrial DNA (mtDNA) alterations reflect cancer-related biological processes, this study investigated whether blood-derived mtDNA profiles could provide clinically relevant information in PC. In this exploratory study, whole-blood mtDNA from 33 PC patients and 10 healthy individuals were analyzed using next-generation sequencing to assess single-nucleotide variants (SNVs), allele frequencies, and mtDNA copy number. A total of 252 unique mtDNA SNVs were identified, including variants exclusive to PC patients, variants unique to controls, and variants shared between groups. While the overall SNV burden did not differ significantly between groups, PC patients showed distinct mutation distributions and allele frequency patterns, with cancer-exclusive variants occurring predominantly at low allele frequencies. Mutation hotspots were observed in the ND5, COI, and D-loop regions, implicating genes involved in oxidative phosphorylation and mtDNA maintenance. Although mtDNA copy number did not differ significantly between groups, greater variability was observed among PC patients and was associated with differences in survival outcomes. Overall, these findings indicate that blood-based mtDNA profiling captures biologically relevant variation associated with PC and supports further development of integrated mtDNA-based approaches for improved risk assessment and patient stratification. Full article

Background: Obesity is a multifactorial chronic disease resulting from sustained energy imbalance and modulated by environmental and demographic factors, and it is associated with numerous comorbidities. DNA methylation is an epigenetic modification associated with obesity. Modulation of DNA methylation is a viable target for obesity control strategies. The flavanol (-)-epicatechin (EC) exerts beneficial effects in overweight individuals, suggesting that EC may influence gene regulation through signaling pathways and epigenetic mechanisms. We evaluated whether EC modulates obesity-associated DNA methylation changes using complementary in silico, in vitro, and in vivo approaches. Methods. In silico analyses were performed to explore potential EC interactions with the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNMT activity was measured in nuclear extracts of 4T1 cells in the presence of EC. Finally, in a C57BL/6 mouse model of diet- induced obesity, we assessed global DNA methylation and the expression of the DNA methyltransferases, as well as metabolism-related genes; peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc-1α), pyruvate dehydrogenase kinase isozyme 4 (Pdk4), and nuclear factor erythroid 2–related factor 2 (Nrf2) and relative mitochondrial DNA content (mtDNA/nDNA ratio) in visceral adipose tissue (VAT) and skeletal muscle. Results. EC showed stable in silico interactions within catalytic/cofactor-binding regions of DNMTs and inhibited DNMT activity in vitro in a concentration-dependent manner. In vivo, the obesogenic diet reduced global DNA methylation and decreased transcript levels of Dnmt1, Dnmt3a, and Dnmt3b in skeletal muscle and adipose tissue. EC counteracted obesity-associated DNA methylation changes in skeletal muscle, restoring global methylation and Dnmt expression toward control levels, whereas effects in VAT were limited. EC increased mitochondrial DNA content. Discussion. In silico and enzymatic data suggest that EC may bind DNMT active sites and inhibit DNMT activity in a concentration-dependent manner, supporting a role for EC in obesity-related epigenetic remodeling, particularly in skeletal muscle. EC also increased relative mitochondrial DNA content in VAT and skeletal muscle despite no obesogenic diet effect on relative mitochondrial abundance, consistent with favorable mitochondrial modulation. In conclusion, EC is an epigenetic modulator and may have positive effects in obesity related dysfunctional tissues. Full article

There is accumulating evidence that distinct mitochondrial DNA (mtDNA) methylation and hydroxymethylation patterns exist in Parkinson’s disease (PD). However, most studies have been limited to the investigation of specific target regions, rather than the entire mtDNA, and have been further hindered by other methodological discrepancies and the lack of non-CpG context investigation. Here, we provide a comprehensive profile of methylation and hydroxymethylation levels across the mitochondrial genome, at global and single-base resolution, in CpG and non-CpG (CHG, CHH) contexts in blood samples from early-onset PD (EOPD) patients (n = 39) and age- and sex-matched controls (n = 63). Bisulfite (BS) and oxidative-bisulfite (oxBS) conversions in parallel workflows followed by next-generation sequencing (NGS) using Illumina’s Novaseq 6000 sequencing system identified mitochondrial 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in all contexts. Global mtDNA methylation was significantly higher in EOPD patients vs. matched controls in the CpG context (p = 5.63 × 10⁻³) in the BS status, and in all contexts [CpG (p = 2.67 × 10⁻⁴), CHG (p = 0.015), CHH (p = 0.012)] in the oxBS status, i.e., “true methylation”. At single-base resolution, the most statistically significant sites across the mitogenome, in the D-loop region, and CpG context, were primarily hypomethylated in EOPD patients compared to matched controls. Upon further validation, both global and base resolution mtDNA (hydroxy)methylation results could act as blood-based biomarkers for EOPD. Full article

Enterobiasis, caused by the nematode Enterobius vermicularis, remains a widespread public health issue, yet data regarding its genetic structure in Southeast Europe are scarce. This study presents the first molecular and phylogenetic characterization of E. vermicularis isolates from Bulgaria. Between 2022 and 2025, perianal tape test samples were collected from 128 individuals (92.2% of whom were children) with enterobiasis from 17 regions of the country. Molecular identification was performed via nested PCR targeting a 324 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, followed by Sanger sequencing. Phylogenetic relationships were analyzed using Maximum Likelihood (IQ-TREE), and population genetic indices were calculated using DnaSP v6. Phylogenetic analysis revealed that all 128 Bulgarian isolates belong to genotype B, clustering closely with sequences from other European and Asian countries. Genetic diversity analysis showed remarkably low variation, with a haplotype diversity (Hd) of 0.1507 ± 0.0416 and a nucleotide diversity (π) of 0.00082 ± 0.00015. Among the 11 identified haplotypes, a single dominant haplotype (Hap_1) accounted for 92.2% of all samples and was distributed across all sampled geographic regions. Tajima’s D was significantly negative (−2.314, < 0.05), suggesting a recent population expansion or purifying selection. The dominance of genotype B and the extremely low genetic diversity suggest a recent introduction or clonal expansion of E. vermicularis in Bulgaria. These findings provide essential baseline data for monitoring transmission dynamics and implementing effective control strategies in the Balkan region. Full article

Alzheimer’s disease (AD), the leading cause of dementia, is expected to markedly increase in prevalence in the coming decades. Beyond amyloid and tau pathologies, accumulating evidence suggests that mitochondrial dysfunction and impaired protein homeostasis play crucial roles in AD onset and progression. Building on our previous identification of molecular signatures associated with disease progression, this study investigated whether epigenetic alterations of mitochondrial DNA (mtDNA) contribute to cognitive decline. Specifically, we analyzed the methylation status of the mtDNA regulatory D-loop region and mtDNA copy number in blood-derived DNA samples from 75 participants who we followed longitudinally over eight years. Subjects were classified into four groups according to clinical progression from healthy cognition to mild cognitive impairment (MCI) and AD. Using a linear mixed-effects model, we observed significant differences in methylation dynamics and mtDNA copy number across groups and time points. Healthy controls showed a progressive increase in D-loop methylation, whereas individuals converting to AD exhibited a marked decrease in its level. An opposite trend was evidenced for mtDNA copy number. These findings suggest that reduced D-loop methylation and increased mtDNA are associated with mitochondrial dysfunction and disease progression, whereas increased methylation may represent a possible protective mechanism. Full article