Oxidase Subunit Research Articles

Prevalence and identification of cyathocotylid trematodes infecting African catfish in Egypt.

The trematode family Cyathocotylidae infects various hosts worldwide, including birds, mammals, reptiles, and fish. However, the lack of molecular data from adult worms hinders phylogenetic, epidemiological, and host association studies. This study aims to identify the common cyathocotylid trematodes infecting African catfish in Egypt using morphological and molecular evidence. Out of 142Clarias gariepinus, 123 fish (86.6%) harbored cyathocotylid metacercariae, with a mean metacercarial intensity of 201 ± 38.5/g. Cyathocotylid metacercariae prevalence gradually rose as host size increased. Although there was no significant difference between groups, larger fish had a higher mean metacercarial intensity. The prevalence and intensity were unrelated to the fish gender. Histopathological examination of metacercariae-infected catfish revealed varying degrees of degenerative changes, including intermuscular edema leading to muscle fiber dispersion and atrophy, involving 11% to over 81% of muscle sections. We identified three cyathocotylid metacercariae and eight cyathocotylid adult species from experimental infection using morphometric and molecular data, including internal transcribed spacer (ITS) and/or mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences. We determined the phylogenetic position of these cyathocotylid samples.The ITS sequence analysis linked the isolated Cyathocotylidae sp. 1 and 2 metacercariae to Prohemistomum vivax adults. Mesostephanus appendiculatoides and Paracoenogonimus ovatus were reported for the first time in Egypt. These findings may provide valuable genetic data for future molecular epidemiological and phylogenetic studies of cyathocotylid trematodes.

Northward migration past the nearctic biogeographical divide; neotropical Gyrodactylus spp. infecting Astyanax have crossed the Trans-Mexican Volcanic Belt

The neotropical fish genus Astyanax (Characidae) and its associated helminths migrated northward from South America following the Great American Biotic Interchange (GABI): ca. 150 Astyanax spp. are found throughout South and Central America, up to the Mexico-USA border. Most characids are distributed south of the Trans-Mexican Volcanic Belt (TMVB), which bisects the country and represents a major transition zone between the neotropical and nearctic realms. Here, we characterize parasites of the monogenean genus Gyrodactylus infecting Astyanax spp. in Mexico: Astyanax aeneus south of the TMBV, Astyanax mexicanus north of it. Based on morphological, phylogenetic (internal transcribed spacer (ITS) and cytochrome oxidase subunit II (cox 2)) and statistical analyses of morphometric data, we confirmed the validity of Gyrodactylus pakan and Gyrodactylus teken, and erected two new species, Gyrodactylus aphaa n. sp. and Gyrodactylus ricardoi n. sp. These four gyrodactylids are part of a complex of morphologically cryptic species, which are phylogenetically closely related to each other, and sister species to Gyrodactylus carolinae and Gyrodactylus heteracanthus, parasites of characins in Brazil. Four gyrodactylid lineages (G. pakan, G. ricardoi n. sp., G. teken, Gyrodactylus sp. A) are distributed north of the TMVB; G. pakan is also widely distributed south of the TMVB, together with G. aphaa n. sp. Based on the ITS phylogeny, Brazilian parasites form a sister clade to all Mexican gyrodactylids, whose derived clades are distributed in progressively more northerly latitudes in Mexico – the three most-derived species north of the TMVB. This would suggest that gyrodactylid species diverged gradually, presumably as their characid fish hosts colonized and adapted to new environments north of the TMVB.

Anthropogenic activities along the Lake Nyasa catchments alter the habitat and genetic diversity of a Lake Salmon, Opsaridium microlepis

The Lake Salmon, Opsaridium microlepis is an economically important fish along the Lake Nyasa. However, the species is under threat of extinction due to anthropogenic activities such as agriculture, mining, urbanization, and deforestation. Consequently, the fish species is currently regarded as a threatened species, and the International Union for Conservation of Nature (IUCN) has red-listed the species due to an apparent decline in abundance. The current study assesses the potential impact of human activities on the genetic diversity of O. microlepis using partial mitochondrial cytochrome oxidase subunit I (COI) sequences and microsatellite loci. The results indicate that genetic diversity is lower in the areas affected by human activities compared to relatively pristine areas. The results from this study may suggest that human activities taking place in the catchments are likely to contribute to the alteration of the genetic diversity of O. microlepis species. Thus, immediate measure is required to control anthropogenic activities in the areas to protect the species and other aquatic organisms from possible threats of extinction.

A NEW SPECIES OF ACANTHOBOTHRIUM (CESTODA: ONCHOPROTEOCEPHALIDEA), PARASITE OF THE GIANT ELECTRIC RAY NARCINE ENTEMEDOR JORDAN AND STARKS, 1895 (BATOIDEA: TORPEDINIFORMES) FROM MEXICO.

Spiral valves from specimens of the giant electric ray Narcine entemedor Jordan & Starks, 1895 were examined in search of tapeworms at 2 localities of the Mexican tropical Pacific Ocean. Acanthobothrium oceguerai n. sp. is described herein based on material from Ventanilla, Oaxaca and from Acapulco Bay, Guerrero. The new species is a category 6 species, distinguished by being apolytic, retaining proglottids on the strobila until they are gravid, having strobila of 166-322 proglottids, having a small scolex and very long bothridia relative to the scolex, and having abaxial prongs that are short and thin in comparison to the axial prongs, which are longer and more robust. Acanthobothrium oceguerai n. sp. can be differentiated from other members of category 6 by the hooks, which are shorter, more robust, and smaller than those of the other members of this category. The phylogenetic analysis based on the 28S rRNA locus placed Acanthobothrium oceguerai n. sp. as sister to an unidentified species of larval Acanthobothrium from Philadelphia, Pennsylvania. In addition, sequences of the mitochondrial cytochrome oxidase subunit I gene and nuclear 18S rDNA were generated to provide support for future taxon sampling. Acanthobothrium oceguerai n. sp. is the fifth species of cestode reported from N. entemedor in the tropical eastern Pacific.

Combating Illegal Trade of Monitor Lizards (Varanus spp) in Malaysia through DNA Barcoding Applications

Abstract Varanids, known as monitor lizards are traded as pet animal commodities and demand for skin trade especially in Southeast Asian countries. Given the significant demand in the international trade, accurate species identification is imperative for Varanids particularly endangered species to prevent misidentification and fraud in marketplaces as Varanids species exhibit morphological similarities. Therefore, advanced molecular technique is vital to facilitate accurate species identification especially for closely related species. Besides, few research have used cytochrome oxidase subunit I (COI) to track the genetic and phylogenetic analysis of Varanids. In this study, the COI mitochondrial DNA region was tested as a molecular marker for species identification of Malaysia monitor lizards using the DNA barcode. Two species were successfully identified from eight samples from Pantai Penyabong, Mersing, Johor which are V. salvator and V. nebulosus based on phylogenetic analysis (Neighbor-Joining, Maximum Parsimony and Bayesian Tree). This study showed that by using their relative genetic divergence and evolutionary links, DNA barcodes can aid in the species identification through molecular technique. Short sequences can be used to identify closely related species, and the possibility of species misidentification among the study’s samples should be taken into account. DNA barcoding techniques should be widely practised especially in wildlife forensics to facilitate precise wildlife identification and improve the efficiencies in combatting illegal wildlife trade in Malaysia.

A new species of planthopper in the genus Eumyndus (Hemiptera: Cixiidae) from palms in eastern Madagascar and molecular evidence for the synonymy of Eumyndus kraussi and Eumyndus metcalfi

A new species of Eumyndus Synave, 1956, a genus endemic to Madagascar, is here described as Eumyndus jeanjacquei sp. nov. The new species was collected from the palm Vonitra fibrosa (C.H.Wright) Becc., 1911. Molecular data are provided for the new species from the barcoding region (5’ half) of the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene and D9-D10 expansion region of the 28S rRNA gene and support placement of the new species in Eumyndus. A new synonymy is proposed based on examination of type material: E. metcalfi Synave, 1956 equals E. kraussi Synave, 1956, new synonym.

Molecular characterization and identification of insect pollinators of Valeriana jatamansi Jones from Shimla Hills, Western Himalaya, India

Valeriana jatamansi Jones a perennial medicinal herb belongs to the family Caprifoliaceae and highly pollinated by wild insect pollinators. In present investigation a total of 51 species of flower visitors were reported on V. jatamansi at altitude range of 1,927 to 2,850 m, in different localities of Shimla Hills, Western Himalaya. The collected insect pollinator species were taxonomically identified and 29 of them were olecularly characterized and identified using the cytochrome c oxidase subunit 1 (COI) sequence from mitochondrial DNA (mtDNA). BLAST analysis revealed 98 to 100 per cent similarity with the existing GenBank sequences. The average AT content was found to be significantly higher (69.8%) compared to the GC content (30.0%), indicating AT bias in all sequences. Phylogenetic analysis of 29 different species of insect ‘pollinator of V. jatamansi revealed two clades, one shows phylogenetic relationship between 28 species which belongs to four orders Diptera, Hymenoptera, Hemiptera and Lepidoptera and the other shows the one species which belongs to order Coleoptera. This study contributes to increasing the number of published accounts of NCBI and helps to accurately distinguish the pollinator fauna of Valeriana Jatamansi.

Updated checklist with new records and molecular data for the mosquitoes (Diptera: Culicidae) of Hong Kong.

An extensive mosquito survey was carried out in Hong Kong from September to October 2022, employing a variety of collection methods. Specimens were identified using a combination of morphology and mitochondrial cytochrome C oxidase subunit 1 (COI) barcode sequences. Twenty-nine species, including three new records, i.e., Culex bicornutus (Theobald), Culex cinctellus Edwards, and Lutzia chiangmaiensis Somboon & Harbach, were collected. Phylogenetic analysis of COI sequences of Culex annulus Theobald and Culex vishnui Theobald collected in Hong Kong and elsewhere revealed that the sequences of the two nominal species are genetically very similar and are included in the same clade. Consequently, the synonymy of Cx. annulus with Cx. vishnui is reinstated. Lutzia halifaxii (Theobald) is removed from the list of species in Hong Kong and is replaced with Lutzia vorax Edwards, the identification of which is confirmed in the present study. The record of Culex spiculothorax Bram recorded in Hong Kong is replaced with the senior synonym Culex sasai Kano, Nitahara & Awaya. The occurrence of Anopheles fluviatilis James and Aedes aegypti (Linnaeus) is discussed. Finally, an updated checklist of the mosquitoes of Hong Kong, which now includes 76 species representing 14 genera, is provided, with notation of those species that vector pathogens of human diseases.

A symbiotic oyster in a shrimp burrow: phylogenetic position of Anomiostrea within the Ostreidae (Bivalvia)

ABSTRACT The ostreid genus Anomiostrea Habe & Kosuge, 1966, is monotypic for A. coralliophila Habe, 1975, which is known as a symbiont inhabiting the burrow of the ghost shrimp Neocallichirus jousseaumei (Nobili, 1904), but despite this unusual habit among oysters its phylogenetic position within the Ostreidae remained unknown. Using specimens collected from two distant localities of the Indo-Pacific, Oman and Japan, we compared shell morphology of these specimens with the holotype and assessed their phylogenetic relationships based on DNA sequence data from two mitochondrial and two nuclear genes. The genetic distance in cytochrome c oxidase subunit I between the specimens from Japan and Oman was 5.5%, indicating substantial geographic differentiation. Our molecular phylogenetic results suggest that A. coralliophila is sister to Ostrea algoensis G. B. Sowerby II, 1871, an oyster from rocky shores in South Africa, and both are closely related to other Ostrea species. This confirms assignment of Anomiostrea to Ostreinae and suggests that this burrow-wall symbiont evolved from typical rocky-shore oysters. Moreover, A. coralliophila was not monophyletic with another symbiotic ostreid Ostrea permollis G. B. Sowerby II, 1871, nor with other symbiotic oysters, indicating that the symbiotic habit evolved multiple times in the Ostreinae.

New Insights on Chromosome Diversification in Malagasy Chameleons

In this work, we performed a preliminary molecular analysis and a comparative cytogenetic study on 5 different species of Malagasy chameleons of the genus Brookesia (B. superciliaris) and Furcifer (F. balteautus, F. petteri, F. major and F. minor). A DNA barcoding analysis was first carried out on the study samples using a fragment of the mitochondrial gene coding for the cytochrome oxidase subunit 1 (COI) in order to assess the taxonomic identity of the available biological material. Subsequently, we performed on the studied individuals a chromosome analysis with standard karyotyping (5% Giemsa solution at pH 7) and sequential C-banding + Giemsa, + CMA3, and + DAPI. The results obtained indicate that the studied species are characterized by a different chromosome number and a variable heterochromatin content and distribution, with or without differentiated sex chromosomes. In particular, B. superciliaris (2n = 36) and F. balteatus (2n = 34) showed a similar karyotype with 6 macro- and 12–11 microchromosome pairs, without differentiated sex chromosomes. In turn, F. petteri, F. major, and F. minor showed a karyotype with a reduced chromosome number (2n = 22–24) and a differentiated sex chromosome system with female heterogamety (ZZ/ZW). Adding our newly generated data to those available from the literature, we highlight that the remarkable chromosomal diversification of the genus Furcifer was likely driven by non-homologous chromosome fusions, including autosome–autosome, Z–autosome, and W–autosome fusions. The results of this process resulted in a progressive reduction in the chromosome number and partially homologous sex chromosomes of different shapes and sizes.

The first detection of the poplar bark aphid, Pterocomma populeum (Hemiptera: Aphididae), in Australia

AbstractPterocomma (Hemiptera: Aphidinae) are large aphids found across the northern hemisphere on various willow (Salix) and poplar (Populus) trees. We provide the first records of the Poplar Bark Aphid, Pterocomma populeum in Australia from Tasmania and New South Wales, a potential pest of poplar trees. Morphological character measurements and DNA (cytochrome oxidase subunit 1) sequences are provided along with discussions around difficulties in species separation.

Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan, Halicephalobus mephisto

In this study, we report a biological temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the complete mitochondrial genome sequence of this organism and show through dN/dS analysis evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively selected amino acid substitutions were located in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase completely shuts down at low temperatures (20 °C), leading to a 4.8-fold reduction in the transmembrane proton gradient (ΔΨm) compared to optimal temperature (37 °C). Direct measurement of oxygen consumption found a corresponding 4.6-fold drop at 20 °C compared to 37 °C. Correspondingly, the lifecycle of H. mephisto takes four times longer at low temperature than at higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success across varying environmental temperatures.

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

BackgroundMosquitoes (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).MethodsA 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.ResultsNovel 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.ConclusionsThis 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.Graphical abstract

WITHDRAWN: First morphological and molecular characterisation of Spirometra mansoni (Cestoda, Diphyllobothriidae) in a domestic cat from Veracruz, Mexico

Cestodes of the genus Spirometra are multi-host parasites that are the causative agents of spirometrosis in domestic and wild carnivores and sparganosis in humans, endemic diseases in tropical and subtropical regions. In domestic animals, the infection is usually asymptomatic or produces gastrointestinal signs such as vomiting and chronic diarrhea. In humans, an incidental parasitosis develops where the larva can lodge in tissues and cause a variety of symptoms, including neuropathies, blindness, paralysis, and death. Described here is a case of spirometrosis in a domestic cat treated in a veterinary clinic in Veracruz, Mexico. The cat presented with an approximate 50 cm section of the strobila protruding from the anus. The proglottids and eggs of the cestode were described via morphological analyses. DNA was obtained from the proglottids for the amplification and sequencing of a fragment of the cytochrome oxidase subunit 1 (cox1) gene. Genetic analysis demonstrated 100 % similarity to Spirometra mansoni. The cat was treated orally with ivermectin/praziquantel (2 mg/50 mg), and the dose was repeated 24 h later. The cat was observed daily for seven days, and no adverse effects occurred. On days 8 and 68 post-treatment, coprological examination revealed no evidence of the parasite. This is the first report of spirometrosis in cats from Mexico.

Endemic Radiation of African Moonfish, Selene dorsalis (Gill 1863), in the Eastern Atlantic: Mitogenomic Characterization and Phylogenetic Implications of Carangids (Teleostei: Carangiformes).

This study offers an in-depth analysis of the mitochondrial genome of Selene dorsalis (Gill 1863), a species native to the Eastern Atlantic Ocean. The circular mitochondrial DNA molecule measures 16,541 base pairs and comprises 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes, and a control region (CR). The nucleotide composition exhibits a notable adenine-thymine (AT) bias, accounting for 53.13%, which aligns with other species in the Carangidae family. Most PCGs initiate with the ATG codon, with the exception of Cytochrome C oxidase subunit I, which starts with GTG. Analysis of relative synonymous codon usage reveals that leucine and serine are the most prevalent amino acids in the mitochondrial genome of S. dorsalis and its congeners (S. vomer and S. setapinnis). All tRNAs display the typical cloverleaf structure, though tRNA Serine (S1) lacks a dihydrouracil arm. Pairwise comparisons of synonymous and nonsynonymous substitutions for all PCGs yielded values below '1', indicating strong purifying selection. The CR spans 847 bp, representing 5.12% of the mitochondrial genome, and is characterized by high AT content (62.81%). It is situated between tRNA-Pro (TGG) and tRNA-Phe (GAA). The CR contains conserved sequence blocks, with CSB-1 being the longest at 22 bp and CSB-D the shortest at 18 bp. Phylogenetic analysis, using Bayesian and Maximum-likelihood trees constructed from concatenated PCGs across 72 species, successfully differentiates S. dorsalis from other carangids. This study also explores how ocean currents and gyres might influence lineage diversification and parapatric speciation of Selene species between the Atlantic and Pacific Oceans. These results highlight the importance of the mitochondrial genome in elucidating the structural organization and evolutionary dynamics of S. dorsalis and its relatives within marine ecosystems.

Relationships between crayfish population genetic diversity, species richness, and abundance within impounded and unimpounded streams in Alabama, USA.

Understanding the relationship between multi-scale processes driving community- and population-level diversity can guide conservation efforts. While the importance of population-level genetic diversity is widely recognized, it is not always assessed for conservation planning, and positive correlations with community-level diversity are sometimes assumed, such that only the latter is measured. We surveyed species richness and cumulative multispecies abundance of crayfishes in impounded and unimpounded streams in the southern Appalachian Mountains (Alabama, USA). We simultaneously assessed levels of population genetic diversity within two focal crayfishes (Faxonius validus and F. erichsonianus) using nuclear (nDNA; inter-simple sequence repeat (ISSR)) and mitochondrial DNA (mtDNA; mitochondrial DNA cytochrome oxidase subunit I (mtCOI)) markers. We then tested for species-genetic diversity correlations (SGDCs), species diversity-abundance correlations (i.e., more individuals hypothesis, MIH), and abundance-genetic diversity correlations (AGDCs) across sites. We also examined the relationship between each of the three different types of correlation (i.e., species richness, cumulative multispecies abundance, and population genetic diversity) and stream habitat characteristics and fragmentation. Surprisingly, based on F. validus mtDNA data, sites with the greatest multispecies abundance had the lowest genetic diversity, indicating a negative AGDC. However, no AGDC was evident from nDNA. There was no evidence of SGDCs for F. validus based on either of the two genetic data types. For F. erichsonianus, there was no evidence for SGDC or AGDC. When considering the community-level data only, there was no support for the MIH. Stream width was positively correlated with F. validus genetic diversity, but negatively correlated with multispecies abundance. Similarly, species richness was positively correlated with stream width in unimpounded streams but negatively correlated with width in impounded streams. These findings indicate that community-level diversity cannot be indiscriminately used as a proxy for population-level diversity without empirically testing this correlation on the focal group. As such, community- and population-level assessments for multiple crayfish species are needed to better understand drivers of diversity and eco-evolutionary processes which will aid in the conservation of this vulnerable taxonomic group.

Fall Armyworm Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) in East Nusa Tenggara Province of Indonesia: Genetic Characterization and Strain Detection

<em>Spodoptera frugiperda</em> J.E. Smith (Lepidoptera: Noctuidae) is an invasive pest of corn plants spreading throughout the world, including East Nusa Tenggara (NTT), Indonesia. Despite the wide distribution, there is a lack of information on the strain or genetic diversity of the pest in NTT. Therefore, this study aimed to determine the strain of <em>S. frugiperda</em> from several areas in NTT with a molecular method using <em>cytochrome oxidase subunit 1</em> (<em>COI</em>)<em> </em>and <em>triose phosphate isomerase</em> (<em>Tpi</em>)<em> </em>gene markers. The<em> </em>samples were collected from 3 islands: Timor, Flores, and Sumba. Amplification of the marker genes was carried out using 3 specific primers to identify the strain obtained from samples. Subsequently, polymerase chain reaction (PCR) products were sequenced and the DNA sequences were analyzed using the BioEdit and BLAST programs. Phylogeny analyses were carried out using the MEGA 11 program to verify the strain group of samples with reference isolates from other countries found in GenBank. The PCR results showed product amplicon size 811 bp for the <em>CO1A</em> marker. Based on phylogeny tree analysis using <em>COI </em>marker, <em>S. frugiperda </em>from NTT showed 2 clades, namely corn and rice strains. The characterization results showed that <em>S. frugiperda</em> in NTT comprised 63.6% corn and 36.4% rice strains. <em>COIB </em>classified <em>S. frugiperda</em> from NTT into the h4 haplotype subgroup, while <em>Tpi </em>gene marker was in the corn strain. This study provided valuable information regarding the strain of <em>S. frugiperda</em> in Indonesia to determine the appropriate control strategy.

Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Ecological connectivity of the Qiongzhou Strait: a case form Orangefin Ponyfish (Photopectoralis bindus) haplotype diversity and genetic structure

Grasping the genetic structure of marine fish populations is vital for comprehending species connectivity patterns and determining the appropriate spatiotemporal scales for conservation management strategies. Here, we analyzed the population genetics of the Orangefin Ponyfish (Photopectoralis bindus Valenciennes, 1835) by examining a portion of the gene coding for the mitochondrial cytochrome c oxidase subunit I. The aim was to evaluate the haplotype pattern, genetic structure, demographic history, as well as the influence of ecological connectivity through the Qiongzhou Strait on the distribution patterns of this species in the northern South China Sea and the Beibu Gulf. In total, 257 specimens yielded only 13 haplotypes, with the predominant haplotype present at all sampling locations. The analysis revealed a “star-like” haplotype pattern, indicating low levels of both haplotype and nucleotide diversity. Additionally, a small but significant genetic structure was observed between the coastal regions flanking the Leizhou Peninsula. These patterns in the haplotype network and genetic structure may be significantly influenced by contemporary currents, particularly through the connectivity of the Qiongzhou Strait. Tajima’s D and Fu’s Fs demonstrated pronouncedly negative values, along with a unimodal mismatch distribution, suggested a recent demographic expansion of Photopectoralis bindus during the late Pleistocene, likely influenced by fluctuations in sea levels.

The genetic legacy of the Quaternary ice ages for West Palearctic butterflies.

The interplay between geographic barriers and climatic oscillations over the past 2.6 million years structured genetic variation at the continental scale. The genetic legacy of the Quaternary ice ages (GLQ) hypothesis outlines this phenomenon for Europe, but a comprehensive data-driven assessment is lacking. Using innovative genetic landscape methods, we model the GLQ in the West Palearctic based on 31,653 Cytochrome c oxidase subunit 1 (COI) sequences from 494 butterfly species and three functional traits. Seven distinct bioregions with varying levels of genetic endemicity emerge, revealing a latitudinal gradient in variation that confirms the "southern richness, northern purity" hypothesis. Through shift from case studies to a comparative approach, we objectively identify the main glacial refugia, colonization routes, and barriers to dispersal. Our findings offer a quantitative model of the GLQ across Europe, North Africa, and neighboring Asia, with broader applicability to other taxa and potentially scalable to encompass life on Earth.