Mitochondrial DNA Research Articles

Detailed characterization of the complete mitochondrial genome of the oceanic whitetip shark Carcharhinus longimanus (Poey, 1861)

BackgroundThe oceanic whitetip shark Carcharhinus longimanus (family Carcharhinidae) is one of the largest sharks inhabiting all tropical and subtropical oceanic regions. Due to their life history traits and mortality attributed to pelagic longline fishing practices, this species is experiencing substantial population decline. Currently, C. longimanus is considered by the IUCN Red List of Threatened Species as “vulnerable” throughout its range and “critically endangered” in the western north Atlantic. This study sequences and describes the complete mitochondrial genome of C. longimanus in detail.Methods and resultsThe mitochondrial genome of C. longimanus was assembled through next-generation sequencing and then analyzed using specialized bioinformatics tools. The circular, double-stranded AT-rich mitogenome of C. longimanus is 16,704 bp long and contains 22 tRNA genes, 2 rRNA genes, 13 protein coding genes and a 1,065 bp long control region (CR). Out of the 22 tRNA genes, only one (tRNA-Ser1) lacked a typical ‘cloverleaf’ secondary structure. The prevalence of TTA (Leu), ATT (Ile) and CTA (Leu) codons in the PCGs likely contributes to the AT-rich nature of this mitogenome. In the CR, ten microsatellites were detected but no tandem repeats were found. Stem-and-loop secondary structures were common along the entire length of the CR. Ka/Ks values estimated for all PCGs were < 1, indicating that all the PCGs experience purifying selection. A phylomitogenomic analysis based on translated PCGs confirms the sister relationship between C. longimanus and C. obscurus. The analysis did not support the monophyly of the genus Carcharhinus.ConclusionsThe assembled mitochondrial genome of this pelagic shark can provide insight into the phylogenetic relationships in the genus Carcharhinus and aid conservation and management efforts in the Central Pacific Ocean.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.

Cryptic Taxa Revealed through Combined Analysis of Chromosomes and DNA Barcodes: The Polyommatus ripartii Species Complex in Armenia and NW Iran.

The detection of cryptic species in complexes that have undergone recent speciation is often difficult, since many standard nuclear markers have not yet accumulated differences between closely related taxa, and differences in mitochondrial markers can be leveled out due to mitochondrial introgressions. In these cases, the use of derived chromosomal characters such as non-ancestral chromosomal numbers and/or unusual karyotype features may be a solution to the species delimitation problem. However, non-ancestral but similar karyotypes may arise secondarily as a result of homoplastic evolution, and their interpretation as homologies may lead to incorrect taxonomic conclusions. In our study, we show that the combined use of mitochondrial DNA barcodes and karyotypes helps to solve this problem and identifies cryptic species in situations where each of these markers does not work individually. Using this approach, we show that the fauna of Armenia and adjacent Iran includes the following cryptic taxa of the Polyommatus ripartii species complex (haploid chromosome number, n in parentheses): P. ripartii paralcestis (n = 90), P. ripartii kalashiani, subsp. nov (n close to 90), P. emmeli, sp. nov. (n = 77-79), P. keleybaricus, sp. nov. (n = 86), P. demavendi belovi (n = 73-75), P. demavendi antonius, subsp. nov. (n = 71-73), P. admetus anatoliensis (n = 79) and P. eriwanensis (n = 29-34). Polyommatus admetus yeranyani is synonymized with P. admetus anatoliensis.

Comparative mitochondrial genome analysis provides new insights into the classification of Modiolinae.

Mitochondrial genomes have become a powerful tool for studying molecular genetics and phylogeny of mollusks. Currently, the position of Modiolinae within Mytilidae and the taxonomic and phylogenetic relationships within Modiolinae were still controversial. This study focuses on the complete mitochondrial genomes of two species: Modiolus modulaides (Röding, 1798) and Modiolus auriculatus Krauss, 1848, which have not been sequenced before. We assembled and characterized the mitochondrial genomes of M. modulaides and M. auriculatus and then analyzed the phylogenetic relationships. The mitochondrial genomes of M. modulaides and M. auriculatus were 15,422bp and 16,027bp, respectively. Both of them were composed of 36 functional genes, including 12 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. All protein-coding genes showed A + T bias, positive GC skews, and negative AT skews in nucleotide composition. Phylogenetic analysis based on the mitochondrial genomes showed that Modiolinae and Bathymodiolinae clustered together to form a sister relationship. Seven Modiolinae species were divided into two clades: L1 (M. modulaides, M. auriculatus and Modiolus philippinarum Hanley, 1843) and L2 [Modiolus modiolus (Linnaeus, 1758), Modiolus kurilensis Bernard, 1983, Modiolus nipponicus (Oyama, 1950), and Modiolus comptus (Sowerby III, 1915)]. The divergence time of the two clades was approximately 105.75Ma. Furthermore, the transfer RNA gene rearrangement, longer genetic distance, and greater genetic differentiation were confirmed between the L1 and L2 clades, as well as differences in the external characteristics of the shells of the two clades. Based on the molecular data, it was speculated that species from the L1 clade might belong to other genera or new genera. This study provides molecular information for further taxonomic and phylogenetic studies of Mytilidae.

The genome sequence of the Cow Parsley Leaf Beetle, Chrysolina oricalcia (O.F. Müller, 1776)

We present a genome assembly from an individual Cow Parsley Leaf Beetle Chrysolina oricalcia (the Cow Parsley Leaf Beetle; Arthropoda; Insecta; Coleoptera; Chrysomelidae). The genome sequence is 1,423.4 megabases in span. Most of the assembly is scaffolded into 22 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.93 kilobases in length. Gene annotation of this assembly on Ensembl identified 35,990 protein coding genes.

Assembly and comparative analysis of the complete mitochondrial genome of Vaccinium carlesii Dunn

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.

Persistence of social structuring in an arid zone mammal following reintroduction

Animal social relationships affect animal survival, reproduction, and resource exploitation, and are important to translocation success, but little is known on how they are impacted in reintroduced populations. Here, we investigate the social genetic structure in a reintroduced population of boodies (Bettongia lesueur), one of few social burrowing marsupial species in Australia. Pairwise relatedness of male and female individuals located within seven warrens was determined using 12 microsatellite markers, while mitochondrial DNA was used to identify maternal ancestral lineage. Females were significantly more related within warrens than between warrens, while the difference among males was not significant. We also found evidence of fine-scale genetic structure up to distances of 2 km in females consistent with patterns found in natural populations. Although mean relatedness values were low, our results support the hypothesis that boodies exhibit a female social structure. Multiple maternal lineages were present within all warrens with one exception, suggesting boodies appear to be non-selective when living with individuals from different ancestral backgrounds. We found a significant positive relationship between female sex bias and the number of active entrances in each warren. Together, these results suggest that social structuring should be a key consideration in future reintroductions of boodies.

IRF1 regulation of ZBP1 links mitochondrial DNA and chondrocyte damage in osteoarthritis

BackgroundZ-DNA binding protein 1 (ZBP1) is a nucleic acid sensor that is involved in multiple inflammatory diseases, but whether and how it contributes to osteoarthritis (OA) are unclear.MethodsCartilage tissues were harvested from patients with OA and a murine model of OA to evaluate ZBP1 expression. Subsequently, the functional role and mechanism of ZBP1 were examined in primary chondrocytes, and the role of ZBP1 in OA was explored in mouse models.ResultsWe showed the upregulation of ZBP1 in articular cartilage originating from OA patients and mice with OA after destabilization of the medial meniscus (DMM) surgery. Specifically, knockdown of ZBP1 alleviated chondrocyte damage and protected mice from DMM-induced OA. Mechanistically, tumor necrosis factor alpha induced ZBP1 overexpression in an interferon regulatory factor 1 (IRF1)-dependent manner and elicited the activation of ZBP1 via mitochondrial DNA (mtDNA) release and ZBP1 binding. The upregulated and activated ZBP1 could interact with receptor-interacting protein kinase 1 and activate the transforming growth factor-beta-activated kinase 1-NF-κB signaling pathway, which led to chondrocyte inflammation and extracellular matrix degradation. Moreover, inhibition of the mtDNA-IRF1-ZBP1 axis with Cyclosporine A, a blocker of mtDNA release, could delay the progression of DMM-induced OA.ConclusionsOur data revealed the pathological role of the mtDNA-IRF1-ZBP1 axis in OA chondrocytes, suggesting that inhibition of this axis could be a viable therapeutic approach for OA.

Mitochondrial DNA depletion syndrome 13. A case report

Mitochondrial DNA depletion syndrome 13. A case report

Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque

BackgroundThe heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition.MethodsWeighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria.ResultsThe CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice.ConclusionsThe results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

The genome sequence of the Sandhill Rustic moth Luperina nickerlii (Freyer, 1845) subspecies leechi Goater, 1976

We present a genome assembly from an individual female Luperina nickerlii (the Sandhill Rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 662.0 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The specimen was confirmed to be a ZO female. The mitochondrial genome has also been assembled and is 15.47 kilobases in length.

The first complete mitochondrial genome of the genus Laelaps with novel gene arrangement reveals extensive rearrangement and phylogenetics in the superfamily Dermanyssoidea.

We collected 56 specimens ofLaelaps chini from the endemic Hengduan Mountain rat species (Eothenomys miletus) and obtained the first complete mitochondrial genome of L. chini by next-generation sequencing (NGS). The L. chini mitogenome is 16,507bp in size and contains 37 genes and a control region of 2380bp in length. The L. chini mitogenome has a high AT content and a compact arrangement with four overlapping regions ranging from 1 to 2bp and 16 spacer regions ranging from 1 to 48bp. We analyzed 13 protein-coding genes of L. chini mitogenome andfound that protein-coding genes in the L. chini mitogenome preferred codons ending in A/U and codon usage pattern was mainly influenced by natural selection. Cox1 has the slowest evolution rate and cox3 has the fastest evolution rate. We combined the mitochondrial genome of eight species ofgamasid mites in the superfamily Dermanyssoidea from Genbank and theL. chini mitochondrial genometo analyze its rearrangement patterns and breakpoint numbers. We found that the L. chini mitogenome showed a novel arrangement pattern and nine species ofgamasid mites in the superfamily Dermanyssoidea, which have been sequenced complete mitochondrial genomes to date, all showed different degrees of rearrangement. Laelaps chini, Echinolaelaps echidninus and Echinolaelaps fukinenensis were closely related species based on genetic distance and phylogenetic analyses. Notably they are clustered with Varroa destructor of the family Varroidae, suggesting that the family Varroidae is more closely related to the family Laelapidae, but more data are needed to test whether Varroa can be classified under the family Laelapidae. The L. chini mitogenome is the first complete mitochondrial genome for the genus Laelaps, and contributes to further exploration of the mitochondrial gene rearrangements and phylogeny for the superfamily Dermanyssoidea.

DNA barcodes are ineffective for species identification of Acropora corals from the aquarium trade.

Species identification of stony corals (Scleractinia), which are regulated under the Convention on International Trade in Endangered Species of Wild Fauna and Flora, is critical for effective control of harvest quotas, enforcement of trade regulations and species conservation in general. DNA barcoding has the potential to enhance species identification success, depending on the specific taxon concerned and genetic markers used. For Acropora, DNA barcoding, based on the mitochondrial putative control region (mtCR) and the nuclear PaxC intron (PaxC), has been commonly used for species identification and delimitation, but the reliability and robustness of these loci remain contentious. Therefore, we sought to verify the applicability of this approach. In this study, we obtained 127 Acropora colonies from the aquarium trade to test the effectiveness of barcoding mtCR and PaxC for species identification. We were able to recover sequences for both loci in over half of the samples (n = 68), while gene amplification and sequencing of mtCR (n = 125) outperformed PaxC (n = 70). Amongst the 68 samples with both loci recovered, just a single sample could be unambiguously identified to species. Preliminary identities, based on only one gene, were assigned for 40 and 65 samples with mtCR and PaxC, respectively. Further analyses of 110 complete mitochondrial genomes obtained from GenBank showed that, despite the full length of the sequences, only eight species were delimited, of which only three species were correspondingly monophyletic. Therefore, we conclude that the commonly used DNA barcoding markers for Acropora are ineffective for accurate species assignments due to limited variability in both markers and even across the entire mitochondrial genome. Therefore, we propose that barcoding markers should generally not be the only means for identifying corals.

PDZK1 protects against mechanical overload-induced chondrocyte senescence and osteoarthritis by targeting mitochondrial function

Mechanical overloading and aging are two essential factors for osteoarthritis (OA) development. Mitochondria have been identified as a mechano-transducer situated between extracellular mechanical signals and chondrocyte biology, but their roles and the associated mechanisms in mechanical stress-associated chondrocyte senescence and OA have not been elucidated. Herein, we found that PDZ domain containing 1 (PDZK1), one of the PDZ proteins, which belongs to the Na+/H+ Exchanger (NHE) regulatory factor family, is a key factor in biomechanically induced mitochondrial dysfunction and chondrocyte senescence during OA progression. PDZK1 is reduced by mechanical overload, and is diminished in the articular cartilage of OA patients, aged mice and OA mice. Pdzk1 knockout in chondrocytes exacerbates mechanical overload-induced cartilage degeneration, whereas intraarticular injection of adeno-associated virus-expressing PDZK1 had a therapeutic effect. Moreover, PDZK1 loss impaired chondrocyte mitochondrial function with accumulated damaged mitochondria, decreased mitochondrion DNA (mtDNA) content and increased reactive oxygen species (ROS) production. PDZK1 supplementation or mitoubiquinone (MitoQ) application alleviated chondrocyte senescence and cartilage degeneration and significantly protected chondrocyte mitochondrial functions. MRNA sequencing in articular cartilage from Pdzk1 knockout mice and controls showed that PDZK1 deficiency in chondrocytes interfered with mitochondrial function through inhibiting Hmgcs2 by increasing its ubiquitination. Our results suggested that PDZK1 deficiency plays a crucial role in mediating excessive mechanical load-induced chondrocyte senescence and is associated with mitochondrial dysfunction. PDZK1 overexpression or preservation of mitochondrial functions by MitoQ might present a new therapeutic approach for mechanical overload-induced OA.

Targeting Mitochondrial Guanine Quadruplexes for Photoactivatable Chemotherapy in Normoxic and Hypoxic Environments.

A first example of a mitochondrial G-quadruplex (mitoG4s) targeted Ru(II) photooxidant complex is reported. The complex, Ru-TAP-PDC3 induces photodamage toward guanine quadruplexes (G4s) located in the mitochondrial genome under hypoxic and normoxic conditions. Ru-TAP-PDC3 shows high affinity for mitoG4s and localises within mitochondria of live HeLa cells. Immunolabelling with anti-G4 antibody, BG4, confirms Ru-TAP-PDC3 associates with G4s within the mitochondria of fixed cells. The complex induces depletion of mtDNA in live cells under irradiation at 405 nm, confirmed by loss of PicoGreen signal from mitochondria. Biochemical studies confirm this process induces apoptosis. The complex shows low dark toxicity and an impressive phototoxicity index (PI) of >89 was determined in Hela under very low intensity irradiation, 5 J/cm2. The phototoxicity is thought to operate through both Type II singlet oxygen and Type III pathways depending on normoxic or hypoxic conditions from live cell imaging and plasmid DNA cleavage. Overall, we demonstrate targeting mitoG4s and mtDNA with a photooxidant is a potent route to achieving apoptosis under hypoxic conditions that can be extended to phototherapy.

Targeting Mitochondrial Guanine Quadruplexes for Photoactivatable Chemotherapy in Normoxic and Hypoxic Environments

A first example of a mitochondrial G‐quadruplex (mitoG4s) targeted Ru(II) photooxidant complex is reported. The complex, Ru‐TAP‐PDC3 induces photodamage toward guanine quadruplexes (G4s) located in the mitochondrial genome under hypoxic and normoxic conditions. Ru‐TAP‐PDC3 shows high affinity for mitoG4s and localises within mitochondria of live HeLa cells. Immunolabelling with anti‐G4 antibody, BG4, confirms Ru‐TAP‐PDC3 associates with G4s within the mitochondria of fixed cells. The complex induces depletion of mtDNA in live cells under irradiation at 405 nm, confirmed by loss of PicoGreen signal from mitochondria. Biochemical studies confirm this process induces apoptosis. The complex shows low dark toxicity and an impressive phototoxicity index (PI) of &gt;89 was determined in Hela under very low intensity irradiation, 5 J/cm2. The phototoxicity is thought to operate through both Type II singlet oxygen and Type III pathways depending on normoxic or hypoxic conditions from live cell imaging and plasmid DNA cleavage. Overall, we demonstrate targeting mitoG4s and mtDNA with a photooxidant is a potent route to achieving apoptosis under hypoxic conditions that can be extended to phototherapy.

The genome sequence of the Mauritius parakeet, Alexandrinus eques (formerly Psittacula eques) (A.Newton &amp; E. Newton, 1876)

We present a genome assembly from an individual male Alexandrinus eques, formerly Psittacula eques (the Mauritius Parakeet; Chordata; Aves; Psittaciformes; Psittacidae). The genome sequence is 1203.8 megabases in span. Most of the assembly is scaffolded into 35 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 18.86 kilobases in length.

The genome sequence of the smooth giant clam, Tridacna derasa Röding, 1798

We present a genome assembly from an individual Tridacna derasa (the smooth giant clam; Mollusca; Bivalvia;Cardiida; Cardiidae). The genome sequence is 1,060.2 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 24.95 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,638 protein coding genes.

Analiza mitohondrijske DNA pri psih pasme šarplaninec in njena uporaba za ugotavljanje filogenetskih povezav med pasmami in znotraj pasme

The Yugoslavian Shepherd Dog – Sharplanina belongs to the group of Molosser type dog breeds and is an autochthonous dog breed in southwestern Kosovo and northwestern North Macedonia. This breed is characterised by its genetic diversity in the mitochondrial DNA. In our research we found nine haplotypes grouped into three main clades A, B and C, with distribution rates of 43 %, 43 % and 14 %, respectively. Our analyses show that the “Sharplanina dog” exhibits a remarkable genetic heterogeneity, which makes it very difficult to determine its origin and to correlate the haplotypes with the geographical location of the collected samples. The geographical proximity of the breed’s origin to the habitat of the extinct ancient Molossian hound and the similarities of its haplotypes with certain dog breeds in Europe and East Asia make it a very interesting breed for further research.

Genetic diversity and population genetics of Schistosoma haematobium isolated from children in Lusaka and Siavonga districts, Zambia.

Urogenital schistosomiasis remains a pervasive health challenge in rural Zambian communities. This study explores the molecular epidemiology and genetic diversity of Schistosoma haematobium using mitochondrial genes (cox1 and nadh1). Urine samples from 421 children in Siavonga and Lusaka districts, Zambia, were collected between December 2020 and February 2022. Microscopy and DNA extraction facilitated the identification of S. haematobium, followed by amplification, sequencing, and phylogenetic analysis of cox1 and nadh1 genes. Phylogenetic analysis revealed clustering with samples from mainland African countries, emphasizing shared haplotypes. Both mitochondrial genes exhibited substantial diversity, with 5 haplotypes from 37 cox1 sequences and 12 haplotypes from 23 nadh1 sequences. High haplotype diversity (0.621-0.808) and low nucleotide diversity (0.00181-0.03288) were observed. Siavonga and Lusaka districts shared the majority of S. haematobium haplotypes. Molecular variance and genetic differentiation analysis indicated variations within populations rather than between populations (cox1: -0.025, nadh1: 0.01646). These findings suggest a limited differentiation between S. haematobium populations in Siavonga and Lusaka, potentially indicating gene flow. Tajima's test revealed negative values, indicating a departure from neutrality, introduction of rare alleles, and recent population expansion. This study contributes essential insights into S. haematobium population genetics, crucial for effective urogenital schistosomiasis control in Zambia.