Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Gut-Liver Axis as a Therapeutic Target for Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a liver disease that remains difficult to predict and diagnose, and the underlying mechanisms are yet to be fully clarified. The gut-liver axis refers to the reciprocal interactions between the gut and the liver, and its homeostasis plays a prominent role in maintaining liver health. It has been recently reported that patients and animals with DILI have a disrupted gut-liver axis, involving altered gut microbiota composition, increased intestinal permeability and lipopolysaccharide translocation, decreased short-chain fatty acids production, and impaired bile acid metabolism homeostasis. The present review will summarize the evidence from both clinical and preclinical studies about the role of the gut-liver axis in the pathogenesis of DILI. Moreover, we will focus attention on the potential therapeutic strategies for DILI based on improving gut-liver axis function, including herbs and phytochemicals, probiotics, fecal microbial transplantation, postbiotics, bile acids, and Farnesoid X receptor agonists.

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

Lipopolysaccharide promotes NLRP3 inflammasome activation by inhibiting TFEB-mediated autophagy in NRK-52E cells.

The NLRP3 inflammasome is involved in many inflammatory diseases. Its activity must be strictly controlled to alleviate the inflammatory process. Autophagy plays a protective role in the negative regulation of NLRP3 inflammasome activation. However, the regulatory mechanism of autophagy controlling NLRP3 inflammasome activation remains to be further investigated. Here, we showed that in NRK-52E cells, lipopolysaccharide (LPS) and ATP stimulation significantly decreased mitochondrial membrane potential, increased ROS production and mtDNA copy number in cytosol. Moreover, autophagic flux was blocked when challenged with LPS and ATP as evidenced by increased LC3 II and p62 expression, reduced TFEB and CTSD expression, and impaired lysosomal acid environment. Furthermore, TFEB deficiency increased cytosolic mtDNA and enhanced LPS and ATP induced NLRP3 inflammasome activation and proinflammatory cytokine expression. Taken together, these findings reveal that LPS and ATP stimulation promoted NLRP3 inflammasome activation through inhibiting TFEB-mediated autophagy in NRK-52E cells, and TFEB could be a potential therapeutic target for the treatment of NLRP3 inflammasome-related kidney diseases.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Maternal Magnolol Supplementation during Pregnancy and Lactation Promotes Antioxidant Capacity, Improves Gut Health, and Alters Gut Microbiota and Metabolites of Weanling Piglets.

Maternal nutrition exerts a profound effect on the postnatal performance of offspring, especially during the weaning period. The multifunctional bioactive component magnolol (MAG) has shown promise as a dietary supplement. This study aimed to explore the effects of maternal MAG supplementation on the antioxidant capacity, gut health, gut microbiome, and metabolome composition of weanling piglets. Fifty pregnant sows were randomly divided into two equally sized groups, the control group and the group supplemented with 100 g/t MAG during the gestation and lactation periods, and 7 days postweaning, the pups were euthanized. The microbiome and metabolome features of weanling piglet colons were compared. Our results revealed that maternal MAG supplementation modified the serum redox status of weanling piglets by decreasing malondialdehyde concentration and increasing superoxide dismutase activity and total antioxidant capacity. Moreover, the decreased indicators of diarrhea were accompanied by improved gut barrier function, in which serum diamine oxidase concentration was decreased, and expressions of zona occludens-1, claudin-1, and intestinal alkaline phosphatase were increased in the colon of weanling piglets from sows supplemented with MAG. Further analysis of the gut microbiota indicated that maternal MAG supplementation significantly increased the relative abundance of beneficial bacteria in the colon of weanling piglets, including Faecalibacterium prausnitzii and Oscillospira. Metabolome analysis identified 540 differential metabolites in the colon of piglets from MAG-fed dams, of which glycerophospholipid classes were highly correlated with progeny gut health and key beneficial bacteria. Our findings indicated that maternal MAG supplementation can improve the oxidative status and gut health of weanling piglets, possibly due to alterations in the gut microbiota and metabolites.

Characterization of the male-specific region containing the candidate sex-determining gene in Amur catfish (Silurus asotus) using third-generation- and pool-sequencing data.

Amur catfish (Silurus asotus) is an ecologically and economically important fish species in Asia. Here, we assembled the female and male Amur catfish genomes, with genome sizes of 757.15 and 755.44 Mb, respectively, at the chromosome level using nanopore and Hi-C technologies. Consistent with the known diploid chromosome count, both genomes contained 29 chromosome-size scaffolds covering 98.80 and 98.73 % of the complete haplotypic assembly with scaffold N50 of 28.87 and 27.29 Mb, respectively. The female (n = 40) and male (n = 40) pools were re-sequenced. Comparative analysis of sequencing and re-sequencing data from both sexes confirmed the presence of an XX/XY sex determination system in Amur catfish and revealed Chr5 as the sex chromosome containing an approximately 400 kb Y-specific region (MSY). Gene annotation revealed a male-specific duplicate of amhr2, namely amhr2y, in MSY, which is male-specific in different wild populations and expressed only in the testes. Amur catfish shared partially syntenic MSY and amhr2y genes with the southern catfish (S. meridionalis, Chr24), which were located on different chromosomes. High sequence divergence between amhr2y and amhr2 and high sequence similarity with amhr2y were observed in both species. These results indicate the common origin of the sex-determining (SD) gene and transition of amhr2y in the two Silurus species. Accumulation of repetitive elements in the MSY of both species may be the main driver of the transition of amhr2y. Overall, our study provides valuable catfish genomic resources. Moreover, determination of amhr2y as the candidate SD gene in Amur catfish provides another example of amhr2 as the SD gene in fish.

Epichaperome inhibition targets TP53-mutant AML and AML stem/progenitor cells.

TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.

The evolutionary patterns, expression profiles, and genetic diversity of expanded genes in barley.

Gene duplication resulting from whole-genome duplication (WGD), small-scale duplication (SSD), or unequal hybridization plays an important role in the expansion of gene families. Gene family expansion can also mediate species formation and adaptive evolution. Barley (Hordeum vulgare) is the world's fourth largest cereal crop, and it contains valuable genetic resources due to its ability to tolerate various types of environmental stress. In this study, 27,438 orthogroups in the genomes of seven Poaceae were identified, and 214 of them were significantly expanded in barley. The evolutionary rates, gene properties, expression profiles, and nucleotide diversity between expanded and non-expanded genes were compared. Expanded genes evolved more rapidly and experienced lower negative selection. Expanded genes, including their exons and introns, were shorter, they had fewer exons, their GC content was lower, and their first exons were longer compared with non-expanded genes. Codon usage bias was also lower for expanded genes than for non-expanded genes; the expression levels of expanded genes were lower than those of non-expanded genes, and the expression of expanded genes showed higher tissue specificity than that of non-expanded genes. Several stress-response-related genes/gene families were identified, and these genes could be used to breed barley plants with greater resistance to environmental stress. Overall, our analysis revealed evolutionary, structural, and functional differences between expanded and non-expanded genes in barley. Additional studies are needed to clarify the functions of the candidate genes identified in our study and evaluate their utility for breeding barley plants with greater stress resistance.

Combined inhibition of BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance of TP53-mutant acute myeloid leukemia to individual BH3 mimetics.

TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53-wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic-driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation.

USC-ENet: a high-efficiency model for the diagnosis of liver tumors combining B-mode ultrasound and clinical data.

Purpose: Ultrasound image acquisition has the advantages of being low cost, rapid, and non-invasive, and it does not produce radiation. Currently, ultrasound is widely used in the diagnosis of liver tumors. However, owing to the complex presentation and diverse features of benign and malignant liver tumors, accurate diagnosis of liver tumors using ultrasound is difficult even for experienced radiologists. In recent years, artificial intelligence-assisted diagnosis has proven to provide effective support to radiologists. However, there is room for further improvement in the existing ultrasound artificial intelligence diagnostic model of liver tumor. First, the image diagnostic model may not fully consider relevant clinical data in the decision-making process. Second, owing to the difficulty in collecting biopsy pathology and physician-labeled ultrasound data of liver tumors, training datasets are usually small, and commonly used large neural networks tend to overfit on small datasets, which seriously affects the generalization of the model. Methods: In this study, we propose a deep learning-assisted diagnosis model called USC-ENet, which integrates B-mode ultrasound features of liver tumors and clinical data of patients, and we design a small neural network specifically for small-scale medical images combined with an attention mechanism. Results and conclusion: Real data from 542 patients with liver tumors (N = 2168 images) are used during model training and validation. Experiments show that USC-ENet can achieve a good classification effect (area under the curve = 0.956, sensitivity = 0.915, and specificity = 0.880) after small-scale data training, and it has certain interpretability, showing good potential for clinical adoption. In conclusion, our model provides not only a reliable second opinion for radiologists but also a reference for junior radiologists who lack clinical experience.

Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo.

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.

Two haplotype-resolved, gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit.

Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value, especially because of the high content of vitamin C in its fruit. In this study, we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var. 'Donghong' (DH) and Actinidia latifolia 'Kuoye' (KY), with total lengths of 608 327 852 and 640 561 626 bp for 29 chromosomes, respectively. With a burst of structural variants involving inversion, translocations, and duplications within 8.39 million years, the metabolite content of DH and KY exhibited differences in saccharides, lignans, and vitamins. A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha, both of which have ultra-high vitamin C content. With each assembly phased into two complete haplotypes, we identified allelic variations between two sets of haplotypes, leading to protein sequence variations in 26 494 and 27 773 gene loci and allele-specific expression of 4687 and 12 238 homozygous gene pairs. Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents; free fatty acids and flavonols accumulated in the early stages, but sugar substances and amino acids accumulated in the late stages. The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit. Compared with wild varieties and other Actinidia species, AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content, providing a possible explanation on why red-flesh kiwifruits are sweeter. Collectively, these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.

Nile tilapia (Oreochromis niloticus) Nanog co-expression with Pou5f3, transcriptional regulation and biological activity in embyonic development and embryonic cells.

Mammalian Nanog is critical in pluripotency acquisition and maintenance. Nonetheless, a recent report from zebrafish (Danio rerio) suggests that Nanog is not required for embryonic cells which is not like the mammalian homologs, but is necessary for the proper formation of the extra-embryonic yolk syncytial layer (YSL). However, whether its biological function in other fishes is conservative remains to be investigated. Our previous work shows that Nanog from Nile tilapia (Oreochromis niloticus) (termed as Ong thereafter) displays differential spatiotemporal expression patterns from the other teleost fishes including zebrafish. In this study, Ong co-expression with Pou5f3 (another core pluripotent transcription factor), transcriptional regulation and its biological functions during embryonic development and in the survival and proliferation of embryonic cells were investigated. At the blastula stage, both Ong and Pou5f3 were highly expressed in embryonic cells and co-located in the nucleus. After that, the expression of both Ong and Pou5f3 began to decrease at the gastrula stage (24 haf) and then exhibited a differential expression profile at the segmentation stage (28-36 haf). Ong disappeared in embryonic cells and was limited to YSL, whilst Pou5f3 was highly expressed in embryonic cells even some with obvious cytoplasmic distribution. Luciferase assay indicated that Ong was negatively regulated by Pou5f3 and positively regulated by androgen and itself. Ong depletion in fertilized one-cell embryos through CRISPR/Cas9 led to blastula blockage or death, and the survival and proliferation of blastula-derived embryonic cells in vitro failed. Collectively, Ong has similar expression and biological function to Pou5f3 at the blastula stage, which is similar to mammalian homolog but different from zebrafish homolog. These data suggest that the expression patterns and functions of Nanog are not conservative in fishes and vary from species to species. This study enriches our understanding about Nanog and its evolution.

Impact of the COVID-19 pandemic on total, sex- and age-specific all-cause mortality in 20 countries worldwide during 2020: results from the C-MOR project.

BACKGROUND: To understand the impact of the COVID-19 pandemic on mortality, this study investigates overall, sex- and age-specific excess all-cause mortality in 20 countries, during 2020. METHODS: Total, sex- and age-specific weekly all-cause mortality for 2015-2020 was collected from national vital statistics databases. Excess mortality for 2020 was calculated by comparing weekly 2020 observed mortality against expected mortality, estimated from historical data (2015-2019) accounting for seasonality, long- and short-term trends. Crude and age-standardized rates were analysed for total and sex-specific mortality. RESULTS: Austria, Brazil, Cyprus, England and Wales, France, Georgia, Israel, Italy, Northern Ireland, Peru, Scotland, Slovenia, Sweden, and the USA displayed substantial excess age-standardized mortality of varying duration during 2020, while Australia, Denmark, Estonia, Mauritius, Norway, and Ukraine did not. In sex-specific analyses, excess mortality was higher in males than females, except for Slovenia (higher in females) and Cyprus (similar in both sexes). Lastly, for most countries substantial excess mortality was only detectable (Austria, Cyprus, Israel, and Slovenia) or was higher (Brazil, England and Wales, France, Georgia, Italy, Northern Ireland, Sweden, Peru and the USA) in the oldest age group investigated. Peru demonstrated substantial excess mortality even in the <45 age group. CONCLUSIONS: This study highlights that excess all-cause mortality during 2020 is context dependent, with specific countries, sex- and age-groups being most affected. As the pandemic continues, tracking excess mortality is important to accurately estimate the true toll of COVID-19, while at the same time investigating the effects of changing contexts, different variants, testing, quarantine, and vaccination strategies.

A new species of Megastigmus (Hymenoptera, Megastigmidae) from China.

Background: Most species of Megastigmus are considered important economic pests that grow in seeds, especially of conifers. Accurate identification of species is a crucial step for the biological research of parasitic pests and the further application of biological control. However, their large variety, small size, similar morphology and different growth and development stages have brought great challenges to taxonomic research. Traditional morphological identification often takes a long time and this requires us to seek a new method for rapid and accurate identification. Therefore, the better identification of Megastigmus urgently needs to be combined with molecular methods to help taxonomic development. New information: Here, Megastigmusdaduheensis sp. n. (Chalcidoidea: Megastigmidae) was identified, based on morphology and molecular markers, such as COI and Cytb. M.daduheensis sp. n. is distinct from other known species of the same genus in the morphology. The results of the molecular phylogenetic tree, similarity alignment and genetic distance indicate that the COI and Cytb sequences of M.daduheensis sp. n. are highly similar to M.sobinae and M.duclouxiana, but there are some genetic differences. The genetic distances of M.daduheensis sp. nov. with M.duclouxiana and M.sabinae were 0.34 and 0.33 and the percentages of shared base pairs were 76.3% and 76.8%, respectively. Both morphological and molecular data classified M.daduheensis sp. n. as a new species. The obtained COI and Cytb sequences of M.daduheensis sp. n. can be used as DNA barcodes, providing molecular data for rapid and accurate identification of this species in the future.