PAK1 variants were described to be causative of neurodevelopmental disorder with macrocephaly, seizures, and speech delay. Herein, we present a de novo PAK1 variant combine with a de novo terminal 1q microdeletion in a Chinese pediatric patient, aiming to provide more insights into the underlying genotype–phenotype relationship.Pathogenic Enrolled in this study was a 6-year-old girl with clinical features of global developmental delay, severe intellectual disability, speech delay, and seizures from Quanzhou region of China. Karyotype and chromosomal microarray analysis (CMA) were performed to detect chromosome abnormalities in this family. Whole exome sequencing (WES) was performed to investigate additional genetic variants in this family.PAK1 gene in the patient, which was interpreted as a likely pathogenic variant.No chromosomal abnormalities were elicited from the entire family by karyotype analysis. Further familial CMA results revealed that the patient had a de novo 2.7-Mb microdeletion  × 1]) in 1q44 region, which contains 14 OMIM genes, but did not overlap the reported smallest region of overlap (SRO) responsible for the clinical features in 1q43q44 deletion syndrome. In addition, WES result demonstrated a de novo NM_002576: c.251C > G (p.T84R) variant in PAK1 variant associated with a terminal 1q microdeletion in a patient with neurodevelopmental disorder. In addition, we believe that the main clinical features may ascribe to the pathogenic variant in PAK1 gene in the patient.In this study, we identify a novel  PAK3 gene have been described in males with X-linked recessive developmental delay (OMIM: 300558) [PAK1 (OMIM: 602590) gene located in 11q13.5q14.1 region is highly expressed during embryogenesis and in adult tissues including the brain, muscle, and spleen [ PAK1 variants were described to be causative of neurodevelopmental disorder with macrocephaly, seizures, and speech delay [The p21-activated kinases (PAKs) are a family of serine/threonine kinases consist of six members PAK1-6), which are active upon Rho GTPases that regulate several signal pathways including Ras/Raf/MEK/ERK and Wnt/β-catenin, and other underlying pathways . PAKs ha 300558) . The PAK, which aZNF238 gene and responsible for corpus callosum abnormalities; the second region contains the AKT3 gene that responsible for microcephaly; the last region is a ~ 100 kb fragment that overlaps HNRNPU, FAM36A and NCRNA00201 genes and is proposed to be the candidate region for seizures [Chromosome terminal 1q microdeletion is a less common chromosome syndrome with only sporadic cases available in the literature, which were commonly identified as interstitial and terminal deletions. Patients with chromosome terminal 1q microdeletion syndrome commonly exhibit developmental delay, intellectual disability, microcephaly, craniofacial anomalies, seizures, and abnormality of the corpus callosum –9. Interseizures .PAK1 gene was identified in the patient using whole exome sequencing.In the study, we present a new de novo terminal 1q microdeletion in a Chinese pediatric patient who manifested developmental delay, speech delay, severe intellectual disability, and seizures, without covering the three distinct SRO in 1q43q44 microdeletion region. In addition, an additional de novo variant in Enrolled in this study was a family from Quanzhou region Fujian province of China. This family denied consanguineous marriage and any familial inherited diseases. Karyotype, chromosomal microarray analysis and whole exome sequencing were carried out for chromosomal abnormalities and genetic variants detection in the family after signed the written inform. Ethics Committee approval was obtained from the Institutional Ethics Committee of Quanzhou Women’s and Children’s Hospital to the commencement of the study (2020No.31).Approximately 2–3 ml peripheral blood were collected from the patient and the parents for karyotype analysis. The peripheral blood lymphocytes were harvested using a SinochromeChromprepII automatic chromosome harvesting system according to the standard protocol , which has been described previously in our study . After swww.qiagen.com).About 3–5 ml peripheral bloods were collected from the patient and the parents for chromosomal microarray analysis, Sanger sequencing and whole exome sequencing. Genomics DNA were extracted from enrolled members’ peripheral blood using QIAamp DNA Blood Kit  according to the manufacturer’s protocol  according to the protocol described previously . Copy nuThe genomics DNA in the enrolled family were further subjected to WES analysis. DNA quantification was carried out using the Qubit dsDNA HS Assay . Approximate mean fragment length of 150–200 bp were sheared using the Covaris LE220 . Then, the sheared DNA were used for library preparation of targeted regions by SureSelect whole-exome capture kit (Agilent). The Illumina DNA Standards and Primer Premix Kit  was used for the sequencing libraries quantification. Subsequently, the Illumina HiSeq 2500 platform  was used for sequencing.The specific process of data analysis was conducted according to the description of our previous study . Data an+4 weeks, with 3.0 kg (+ 0.2 SD) in birth weight and a 50 cm (− 0.6 SD) in height. No threatened abortion or prenatal ultrasound anomalies were observed during the pregnancy. However, an obvious developmental milestone delay was observed, she was unable to sit independently at 18 months, could crawl at 26 months, and walk independently at over 4 years of age. A subsequent children psychological test elicited a low intelligence quotient (scores: 19), based on which a diagnosis of severe intellectual disability was made.Recruited in this case report was a 6-year-old girl, who was the first child of the family. Both of her parents were 30-year-old, who denied consanguineous marriage and any history of familial genetic diseases. She was born naturally at the gestational age of 39+ years, seizures occurred, accompanied with loss of consciousness, clenched fists, twitching limbs, without foaming at the mouth and fever. Seizures could relieved spontaneously in about one minute, with a daily frequency of 4–5 episodes. The symptom could be controlled by oral administration of antiepileptic drugs. Electroencephalography (EEG) showed abnormal brain waves, including synchronous paroxysmal slow wave rhythm with high amplitude, asymmetric amplitude on both sides, and overlapping sharp slow waves during the period, which was obvious in both frontotemporal areas. No abnormalities were observed in brain MRI detection. She is now 6 years and 10 months old with 115 cm (− 1.4 SD) in height and 23 kg (+ 0.2 SD) in weight. But she is unable to speak and defecate by herself, and she could only understand simple instructions. In addition, physical examination showed that she had normal consciousness, and limb muscle strength was defined as class V. She had a normal head circumference, with no significant deformities in the hands and feet, but mild facial abnormalities were observable including ocular hypertelorism, flat nasal bridge, irregular teeth, and hydrostomia. Later, the couple gave birth to a girl and a boy in 2018 and 2021, respectively, both showing normal clinical features and developmental milestones.At the age of 3SMYD3, TFB2M, CNST, AHCTF1, ZNF695, ZNF124, ZNF496, NLRP3, OR13G1, OR2W3, OR2M7, OR14I1, LYPD8, and ZNF692  × 1) in 1q44 region, containing 14 OMIM genes including PAK1 gene in the patient and verified by Sanger sequencing  the c.251C > G variant was predict to affect protein structure/function (Damaging scroe: 0.83) (PP3). In addition, the c.251C > G variant in PAK1 gene located in the p21-Rho-binding domain, which binding Cdc42p- and/or Rho-like small GTPases according to the UCSC database (PM1). Furthermore, the patient’s clinical presentation is consistent with this gene evaluated by clinical experts (PP4). Finally, the variant was interpreted as likely pathogenic variant according to the ACMG guidelines .WES technology was further employed to investigate additional variants in the patient using peripheral blood. A novel NM_002576: c.251C > G (p.T84R) variant in  PAK1 gene variant associated with a de novo terminal 1q44 microdeletion, without covering the reported three distinct SRO in 1q43q44 microdeletion region.In the clinical practice, CMA technology manifests a great advantage in copy number variants (CNVs) detection, as well as uniparental diploid and triploid, and it has been recommended as a first-line detection tool in etiological diagnosis of patients with multiple congenital anomalies , 27. In PAK1 gene were related to intellectual developmental disorder with macrocephaly, seizures, and speech delay . To date, extremely rare reports of PAK1 variants that result in IDDMSSD are available in the literature. Additionally, most of the patients had de novo PAK1 variants [CDC42  and RAC1 . In addition, pathogenic variants in RAC1 and CDC42 genes are associated with developmental disorders [PAK1 gene exhibited developmental delay, macrocephaly, seizures, and ataxic gait [PAK1 variant was also supported that knockout of either PAK1 or PAK3 in mice results in no obvious abnormalities [PAK1 gene, indicating the important role of PAK1 in controlling postnatal brain development and volume. However, a previous study conducted by Horn et al. [PAK1 gene variants with intellectual disability, macrocephaly and seizures, with one of them did not manifests macrocephaly.Causative variants in IDDMSSD; 18,158. Tisorders . A previIDDMSSD; 18,158. Tmalities . In addimalities  present n et al.  presentePAK1 gene was also identified using WES technology who had similar features including global developmental delay, severe intellectual disability, speech delay, and seizures, without macrocephaly. Thus, the clinical feature of macrocephaly may manifest incomplete penetrance in patients with PAK1 variants. As for the importance of PAK1 in neuronal growth and structure, as well as the previous reported cases, we believe that the de novo PAK1 gene variant in the patient may responsible for the major clinical features of neurodevelopmental disorder. However, we can not rule out the pathogenic of 1q44 microdeletion that contributing to the phenotypes such as developmental delay and intellectual disability.In our case report, a de novo c.251C > G (p.T84R) variant in SMYD3 were contained. As demonstrated in the DECIPHER database, two cases with 1q44 microdeletion (DECIPHER ID: 338648 and 426113) only containing SMYD3 gene exhibited global developmental delay, intellectual disability, seizures, and stereotypy. SMYD3 is a histone methyltransferase, playing a role in transcriptional regulation as a RNA polymerase complex, and also an important role in carcinogenesis and metastasis [SMYD3 was responsible for the intellectual disability phenotype in their cases. Thus, the SMYD3 deletion may also responsible for partial clinical features in this study.Although the terminal 1q microdeletion in our study did not covering the reported three distinct SRO in 1q43q44 microdeletion region, several OMIM genes including tastasis  indicatePAK1 gene was identified, which may be the main reason for the patient’s clinical phenotypes. Moreover, our study also strengthened the application value of CMA and WES in the etiologic diagnosis of in patients with unexplained congenital abnormality.In conclusion, our study presented a patient with terminal 1q microdeletion with global developmental delay, severe intellectual disability, speech delay, and seizures, without covering the reported three distinct SRO in 1q43q44 microdeletion region. Interestingly, an additional c.251C > G (p.T84R) variant in


Most bacteria attach to biotic or abiotic surfaces and are embedded in a complex matrix which is known as biofilm. Biofilm formation is especially worrisome in clinical settings as it hinders the treatment of infections with antibiotics due to the facilitated acquisition of antibiotic resistance genes (ARGs). Environmental settings are now considered as pivotal for driving biofilm formation, biofilm-mediated antibiotic resistance development and dissemination. Several studies have demonstrated that environmental biofilms can be hotspots for the dissemination of ARGs. These genes can be encoded on mobile genetic elements (MGEs) such as conjugative and mobilizable plasmids or integrative and conjugative elements (ICEs). ARGs can be rapidly transferred through horizontal gene transfer (HGT) which has been shown to occur more frequently in biofilms than in planktonic cultures. Biofilm models are promising tools to mimic natural biofilms to study the dissemination of ARGs via HGT. This review summarizes the state-of-the-art of biofilm studies and the techniques that visualize the three main HGT mechanisms in biofilms: transformation, transduction, and conjugation. Up to 80% of all bacteria mainly live in biofilm communities ,2. The nAlthough biofilms can be beneficial for gut homeostasis and food digestion , environThis review presents the state-of-the-art of the literature on HGT in biofilms. We aimed to show HGT studies of both Gram-positive and Gram-negative bacteria in biofilms. However, research on conjugative plasmids and ICEs from Gram-positive bacteria has been highlighted, as it has not been reviewed as extensively as works from Gram-negative bacteria. Furthermore, we wanted to point out the drawbacks and restrictions of HGT studies in biofilm communities.PubMed and Google scholar were used as electronic databases for the literature search.in situ model”, “in vitro model”, “in vivo model”, “integrative and mobilizable elements”, “integrative conjugative elements”, “mating”, “medical device”, “membrane vesicle”, “microfluidic”, “monomicrobial”, “mono-species”, “multi-drug resistance”, “multi-species”, “nosocomial infection”, “outer membrane vesicle”, “pathogen”, “phage“, “phage therapy”, “planktonic culture”, “plasmid”, “polymicrobial”; “T4SS”, “transduction”, “transformation”, “transposon” or “wastewater treatment plant”.The keywords used in the bibliographic research were: “abiotic factor”, “antibiofilm”, “antibiotic resistance”, “biofilm”, “biofilm formation”, “biofilm formers”, “biofilm study”, “biofilm-associated infection”, “biotic factor”, “CLSM”, “conjugation”, “conjugative plasmid”, “device-related infection”, “environment”, environmental factor”, “horizontal gene transfer”, “hospital”, “hot spot”, “ICE”, “IME”, “implant”, “The selection of articles is based on the following main criteria: (1) experimental evidence for HGT and (2) “real” biofilm studies.The multistep biofilm formation process has been studied in great detail. It starts with the adherence of planktonic cells to a surface and EPS formation, followed by microcolony formation, differentiation into a mature biofilm and partial detachment and dispersion for new surface colonization ,20,21. Tin vitro Pseudomonas aeruginosa biofilms and has been widely generalized to describe all biofilms .Beaudoin et al. demonstrated that the conjugative transfer rate can be determined in situ . After bP. putida donor cells carrying a reporter plasmid whose fluorescence was repressed by a chromosomal repressor (lacIq). Therefore, only transconjugant cells were fluorescent [In situ determination of conjugative transfer rates in defined biofilms with GFP labelling was performed by Hausner and Wuertz [CLSM can be used as a non-invasive technology to directly monitor HGT in biofilms ,304,305.orescent ,305,307.d Wuertz . Transfein situ is a dual-label monitoring tool that uses fluorescent reporter genes without any additional stains [dsRed gene) and the conjugative TOL plasmid (gfp gene) for in situ monitoring of plasmid transfer in biofilm reactors [gfp. The GFP expression was controlled by a lacIq-repressible promoter and LacI production in the donor [lacIq-repressible promoter in the recipients, gfp expression was restored. In this study, E. coli, P. putida and Kluyvera sp. strains were used as donors and a soil microbial community as recipients. Thus, the differentiation of donor, recipient, and transconjugant cells is highly specific.An optimized technique for studying conjugation l stains ,308. Thireactors ,304,309.E. coli were subjected to varying acidic pH values and simultaneously used for the anaerobic expression of fluorescent proteins mCherry and GFPmut3 [There exist several challenges when applying the presented methods to biofilms: Bacteria within natural biofilms underlie local gradients of abiotic factors  . GFP req GFPmut3 . The fluin situ biofilm formation [E. coli donor strain carried the GFP-encoding IncP-1 plasmid pKJK5 with dual-label fluorescence [A promising platform for studying HGT in biofilms is microfluidics. The technique allows on-chip mating assays for plasmid-mediated conjugation in biofilms at a microscale level. This method enables long-term cultivation, time-lapse and real-time tracking of transfer processes, transconjugant formation even at the single-cell level, and real-time imaging of ormation ,313. In rescence ,294,314.rescence ,294,314.Fluorescent labeling methods can be combined to specifically detect donors, recipients and transconjugants. In contrast to in vitro HGT determination, time-lapse imaging using microfluidics can further distinguish between vertical transmission and HGT. Randomly appearing GFP spots can be described as primary HGT events, the fluorescence extension from these spots is due to cell division .gfp expression by the natural lac operon in E. coli recipients [However, there are also some drawbacks of the system: potential instability of GFP-encoding plasmids in transconjugants or repression of cipients  or autofcipients  cannot bICEs, formerly termed conjugative transposons, are MGEs that encode genes for their own excision, conjugation, and integration . ICEs arAs IMEs are rarely described, no HGT studies in biofilms exist to date.att) sites [ICEs can exist in circular extrachromosomal forms upon excision and transfer to a new host . However, they are not stable as an extrachromosomal intermediate. They replicate only when integrated into the host chromosome via site-specific recombination between defined attachment (t) sites . The ICEt) sites ,323,324.t) sites ,324,325.There is little knowledge on ICEs compared to plasmids. A review from 2020 compared PubMed searches with combined keywords such as “antibiotic resistances” AND “integrative conjugative element” or AND “conjugative transposon” with that of “plasmid” AND “antibiotic resistance” . Only 2–B. subtilis ICEBs1 [V. cholerae ICESXT-R391 [E. faecalis Tn916 [, Bacteroides thetaiotaomicron CTnDOT [Pseudomonas knackmussii ICEclc, Streptococcus thermophilus ICESt1/3 [Mesorhizobium loti ICEMlSymR7A [St1 and ICESt3 [Bs1 [Evolutionary dynamics and the mechanism of ICE conjugation remain largely unexplored . Althougs ICEBs1 , V. cholSXT-R391 , E. faecis Tn916 , Bacteron CTnDOT , PseudomICESt1/3 ,336, andMlSymR7A . The genMlSymR7A . To imprd ICESt3  as well St3 [Bs1 ,339.Bs1. They were compared with solid surface matings. The donor–recipient mixture was incubated on biofilm-stimulating growth medium to induce biofilm formation. The same experimental setting has been used in several ICEBs1 studies [Bs1 transfer frequency was increased by extracellular matrix production by the recipient cells.Conjugation studies in biofilms have only been performed with ICE studies ,339,340.B. subtilis biofilms was analyzed by cryomicrotome and fluorescence microscopy [Bs1 transfer rates were almost exclusively driven by transconjugants as secondary transfer events. HGT was mainly mediated by transconjugants in close vicinity to initial donor cells, in so-called conjugative clusters, located close to the air–biofilm interface.ICE dissemination in croscopy . High ICBs1 T4SS machinery. The knockout was only complemented in trans in the donor. Thus, transconjugants cannot transfer ICEs via conjugation.It is often not possible to determine exactly the impact of vertical transfer on the dissemination of MGEs within the biofilm. To this end, Bourassa et al.  deleted Bs1 transfer in B. subtilis cells was visualized in real time by fluorescence microscopy [gfp gene to the E. coli Lac repressor lacIq (LacI-GFP) inserted into ICEBs1. Thus, B. subtilis cells which had acquired ICEBs1 showed green fluorescence. B. subtilis cells grow naturally in chains. The authors demonstrated that rapid ICEBs1 spread among cells within the bacterial cell chain occurred via ICE conjugation.As in the conjugation experiments of Klümper et al. , fluorescroscopy . Red fluTo follow ICE transfer in real time, this fluorescent method could be applied to biofilm models, such as flow chambers or microfluidics. In comparison to plasmids, more extensive studies are needed to better validate the role of ICEs in biofilms.in situ techniques such as CLSM or microfluidics would immensely enhance the knowledge of gene spread in complex environments. The use of modern and more accurate methods such as Hi-C or CRISPR–Cas spacer acquisition can help overcome the insufficient linkage of MGEs to their hosts and the lack of detection of transient HGT events. In addition, coupling biofilm studies with the application of modern HGT detection and quantification methods would help identify risk scenarios to develop intervention strategies against ARG spread.This review aimed to provide deeper insights into the ability of pathogens to acquire ARGs and adapt to changing environments. Environmental and clinical biofilms are reservoirs of ARG-encoding bacteria. They are a serious threat to public health, as they can intervene with the treatment of infectious diseases. HGT mechanisms, such as transformation, transduction, and conjugation, shape the genetic diversity of bacteria and contribute to their rapid adaptive evolution. Mimicking biofilm conditions facilitates studying the spread of ARGs across different ecological systems. HGT experiments in biofilms are still underrepresented as biofilms are highly complex structures that are difficult to mimic under laboratory conditions. Thus, multiple approaches are required to study horizontal gene spread in biofilms. Most biofilm studies still use invasive methods to determine HGT rates. Coupling classical methods with The pool of MGEs encoding ARGs is immense. However, the transfer features of OMVs, MVs, ICEs, IMEs, integrons and other mobile gene cassettes are not as well characterized as those of conjugative plasmids. The dissemination potential of these mobile elements in heterogeneous environments such as biofilms should not be underestimated and should be studied to a greater degree in the future.In summary, biofilms should be an important target for regular monitoring and control of the occurrence and dissemination of ARGs in clinical settings, industry, and the environment.


Macquartia belongs to Diptera, Tachinidae, and Tachininae, among which there are 12 recorded species. Whilst previous research on Macquartia has focused on the description of species morphology, a few studies have explored the phylogenetic relationships, but with a restricted number of species and several DNA fragments, which are insufficient to study the phylogeny of a genus, especially in order to understand the evolution of ecological traits in Tachinidae. To provide further insight into the relationships of Macquartia, the complete mitochondrial genomes of M. brunneisquama sp. nov., M. chinensis sp. nov., M. flavifemorata sp. nov., and M. flavipedicel sp. nov. were sequenced and compared in this study. The mitogenomes of these four Macquartia species were discovered to be highly conserved. Phylogenetic analyses, based on the mitogenome data of 19 species of Tachinidae and outgroups, supported the monophyly of Macquartia and generated basic data for the phylogenetic study of the Tachinidae.The subfamily Tachininae comprises over 2700 species on Earth, making it the second largest group of Tachinidae, some of which have the potential to be used in the biocontrol of agricultural and forest pests. This subfamily shows the largest morphological variance, which is reflected by their great diversity in terms of ecology and host preferences. Macquartia Robineau-Desvoidy  represents one of the most ancient evolutionary lineages of tachinids, parasitizing Chrysomelidae larvae. We found four new Macquartia species collected by malaise traps, namely M. brunneisquama sp. nov., M. chinensis sp. nov., M. flavifemorata sp. nov., and M. flavipedicel sp. nov. These new species are described and illustrated, and their comparison with congeners as well as an identification key to the 12 species of Macquartia from China known to date are included. To determine the significance of the mitogenome architecture and evolution across different tachinid lineages of this primitive taxonomic group, four complete mitochondrial genomes were sequenced, annotated, and analyzed. The gene arrangements are consistent with the ancestral insect mitogenomes. The full-length sequences and protein-coding genes (PCGs) of the mitogenomes of the four species are all AT-biased. Analyses of Ka/Ks and overall p-genetic distance demonstrated that nad5 showed the highest evolutionary rate and nad1/nad4L were the most conserved genes among the four species. Phylogenetic reconstruction based on 13 PCGs strongly supported the monophyly of Macquartia, and the relationships of the four species are (M. flavifemorata + (M. flavipedicel + (M. brunneisquama + M. chinensis))). This study will help enhance our understanding of the taxonomic status and phylogenetic relationships in Tachinidae. Macquartia Robineau-Desvoidy (Diptera: Tachinidae) is a genus from the tribe Macquartiini, with 25 species that are widely distributed in the Afrotropical, Oriental, Palearctic and Nearctic regions, and counting eight species recorded in China [Tachinidae are one of the most morphologically diverse yet evolutionarily recent families of Diptera with 1248 described species in China [in China .Macquartia with a known biology are parasitoids of Coleoptera larvae dwelling in Chrysomelidae [Macquartia has a close phylogenetic relationship with Porphyromus and Myiophasiini  based on molecular evidence. Herting and Dely-Draskovits synonymized many genera from the Palearctic Region under Macquartia. Different systematic treatments of Macquartia are mainly due to morphological similarity among the genera of Macquartia and Dufouria, Melastrongygaster, Gnadochaeta, Porphyromus, and Myiophasiini, as well as to a lack of molecular evidence in 1993 [Macquartia and characterize the genus with a new generic definition [Billaea, Dexiinae) to Chrysomelidae .Species of omelidae . Cerrettomelidae . Stiremaomelidae . Under t in 1993 . We follfinition ,9,10. Thfinition . We founMacquartia have been released on the NCBI. The lack of mitogenomic data has limited our understanding of the evolution of Tachinidae. Here, we document the mitogenomic data of Macquartia and reconstruct the phylogenetic relationships of the family Tachinidae with newly sequenced mitogenomes to broaden our understanding of its mitogenomic structures and phylogenetic relationships. In addition, 287 specimens of Macquartia from China were examined, and 4 new species of Macquartia from China were discovered and described; a key to 12 species from China is given based on the examined material and related data. Hopefully, this study will promote future research into the taxonomy and evolutionary development of Tachinidae.The mitochondrial genome exhibiting the characteristics of a small size, matrilineal inheritance, and high copy numbers has become a powerful marker for specimen identification, phylogenetic relationships, and comparative genomics analysis. To date, only two mitogenomes in this subfamily have been sequenced and assembled, and no mitogenomes of Macquartia were morphologically identified based upon morphological characteristics, especially male genitalia, by the corresponding author. The morphological terminology used in the descriptions follows McAlpine [The collected information on tachinid specimens in this study is shown in . Four neMcAlpine , except McAlpine . SpecimeMcAlpine . DissectMcAlpine  and the cox1 [cox1 as bait sequences [http://mitos2.bioinf.uni-leipzig.de/index.py) (accessed on 8 February 2022) [Macquartia were uploaded to GenBank with the accession numbers of OL681848-OL681851.Total genomic DNA was extracted from a single fly using the DNeasy Blood and Tissue kit  following the manufacturer’s instructions for the total DNA purification of animal tissues. The little genomic DNA was used for the PCR amplification and sequencing to obtain DNA sequences of cox1 ; subsequcox1  and acqucox1  with coxequences ,19. Prothttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html) (accessed on 21 February 2022) [Macquartia mitogenomes. The ratios of Ka (nonsynonymous substitutions)/Ks (synonymous substitutions) based on 13 aligned protein-coding genes were estimated with DnaSP v6 [Macquartia species under the Kimura 2-parameter mode [The circular mitogenome maps were depicted using OGDRAW (ry 2022) . The sliDnaSP v6 . MEGA 5 ter mode .Lucilia sericata  and Sarcophaga crassipalpis (Sarcophagidae) were chosen as outgroups. Phylogenetic analysis was performed on the dataset of 13 PCGs, where all the assembled PCGs of 19 mitogenomes were aligned through MAFFT [Mitochondrial genomes of 19 species representing all four subfamilies of Tachinidae were selected for phylogeny reconstruction, including the four new mitogenomes documented in the present study. gh MAFFT . The optgh MAFFT . The Baygh MAFFT . The BI gh MAFFT ,30.Macquartia Robineau-Desvoidy, 1830.Genus Macquartia Robineau-Desvoidy, 1830: 204. Type species: Macquartia rubripes Robineau-Desvoidy, 1830 .Remarks: We follow Herting and Dely-Drskovits (1993) [Macquartia. For practical reasons, and to reduce the amount of changes in traditional binominal usages.s 1993) , Tschors93 , Tschs (1993) , Chao ets (1993) , O’Hara Generic diagnosis. Macquartia may be easily distinguished from other tachinids by the following characters: hairs or setulae on posteroventral half of head all black, without any pale hair. Second costal section with fine hairs ventrally. Scutum with three pairs each of presutural dorsocentral bristles and postsutural intra-alar bristles. Preapical posteroventral seta on hind tibia distinctly shorter than the preapical anteroventral seta.Description. Body length more than 4.0 mm. Eye densely covered with hairs, each hair longer than combined diameter of three eye facets. Frons of male at most one-fourth eye width in dorsal view, proclinate orbital setae absent in male, or with two proclinate orbital setae in female. Ocellar setae developed, proclinate. Lower margin of face usually not visible in profile. Facial ridge with setae on lower half or less. Hairs or setulae on posteroventral half of head all black, without pale hairs. First flagellomere not pointed apically, less than two times as long as pedicel. Arista thickened on basal one-fourth or less, first aristomere distinctly shorter, arista short pubescent, the longest hairs longer than basal diameter of arista or plumose. Palpus dark brown or at least reddish yellow at apex. Scutellum and abdomen black. Scutum before suture without dark stripes, with two or three pairs of presutural and usually three postsutural dorsocentral setae, two or three postsutural intra-alar setae present. Scutellum usually with three pairs of setae along its margin, with strong crossed apical setae, but weaker than subapical scutellar setae, lateral scutellar setae absent or present. Prosternum and proepisternum bare. Postpronotum with two or three setae, arranged in a straight line. Katepisternum with two or three setae, anepimeral setae well developed, anatergite almost always with a group of minute hairs or setulae below lower calypter. Second costal section of wing with fine hairs ventrally. Only base of vein R4+5 with hairs. Wing cell r4+5 open or with a very short petiole, Bend of vein M without continuation or with a very short stub. Dorsal surface of lower calypter bare, divergent from scutellum or not divergent from scutellum. Legs usually black, sometimes reddish yellow. Inner anterior surface of fore coxa bare or predominantly bare. Preapicall posteroventral seta on hind tibia distinctly shorter than preapical anteroventral seta. Middorsal depression on abdominal syntergite 1 + 2 not or extending back to the hind margin of that segment, without or with two median marginal setae, fourth tergite barely with a complete row of discal setae. Male genitalia very long, middle part of cerci narrowed and pointed, surstyli more or less narrowed.MacquartiaKey to Chinese species of 1-Middorsal excavation of abdominal syntergite 1 + 2 not extending to its posterior margin. Three postsutural dorsocentral setae. Palpi dark brown or reddish yellow…………………………………………………………………………………..5Middorsal excavation of abdominal syntergite 1 + 2 extending or nearly extending to its posterior margin. Three or four postsutural dorsocentral setae. Palpi reddish yellow.…………………………………………………………………………………….....22-M. tessellum MeigenParafacial hairy. Arista almost bare; four postsutural dorsocentral setae; three katepisternal setae. Lower calyptrae not divergent from scutellum. Abdomen black, with gray pruinosity and markings; tergite three with large black marking………………………………………………………….Parafacial bare, at most with 3–4 hairs below lowest frontal seta. Aristal hairs at least more than diameter of aristal base. Four or three postsutural dorsocentral setae. Two katepisternal setae. Lower calyptrae divergent from scutellum. Thoracic scutum with gray pruinosity, abdomen black, with sparse pruinosity or without pruinosity, syntergite 1 + 2 with a pair of lateral marginal seta, without median marginal seta; third tergite with two median marginal setae, fourth tergite with a row of marginal setae……………………………………………………………………………………….…3 3M. pubiceps Zetterstedt-Pedicel and basal half of flagellomere yellow. Three postsutural dorsocentral setae. Third and fouth tergites without median discal setae in both sexes, ocelli yellow.…………………………………………………………………………………4Pedicel and basal half of flagellomere dark brown. Four postsutural dorsocentral setae. Basicosta reddish yellow. Third tergite with two median discal setae in male and absent in female, fourth tergite with two to a row of discal setae in male and two setae in female, ocelli red …………………………………………………………...4M. flavipedicel Zhang and Li sp. nov.-M. flavifemorata Zhang and Li sp. nov.Basicosta and legs reddish yellow. Eyes with densely long hairs in male and sparsely short hairs in female. ………………Basicosta dark brown. Legs black. …………………5-Mid tibia with 2–5 anterodorsal setae. Abdominal third tergite with 2–4 marginal setae………………………………………………………………………………….....7Mid tibia with one anterodorsal seta. Lower calyptrae divergent from scutellum……...……………………………………………………………………………6 6M. chinensis Zhang and Li sp. nov.-M. macularis VilleneuveParafacial hairy only on upper half or hairy on whole length ……………………………………………….………………Parafacial bare, at most with 3–4 hairs below lowest frontal seta. Legs black. syntergite 1 + 2 without median marginal and with 1–2 lateral marginal setae ……………………………………………………………7-Parafacial bare, at most hairy on upper half..…………………………...……….....9Parafacial hairy on whole length …………………………………………..……………...8 8M. dispar Fallén-M. viridana R.-D.Pedicel and tibiae at least reddish yellow. Femora at least reddish yellow on ventral apex. Two katepisternal setae. Frons width at least 1/4 of eye width, frontal vitta about twice as wide as parafrontalia. Prealar seta about as long as hind supra-alar seta in famale. Abdomen covered with dense grayish pruinosity and regular gleaming marking…………………………………………Pedicel and legs black ………………………………………………..……9-Lower calyptrae divergent from scutellum..………………………………………11Lower calyptrae not divergent from scutellum. Palpi reddish yellow, seldom dark at apex…………………………………………………………………………………………1010M. tenebricosa Meigen-M. chalconota MeigenHind tibia with two preapical dorsal setae; syntergite 1 + 2 without median marginal seta. Abdomen of female covered with covered grayish pruinosity, with gleaming markings ………………………………………Hind tibia with three preapical dorsal setae, middle one weaker; three katepisternal setae. Frons 1/7–1/8 of eye width, frontal vitta narrower than parafrontalia. Parafacial hairy on upper half. Abdomen with sparse grayish pruinosity in male and female, or female black, without pruinosity, syntergite 1 + 2 with 2 median marginal and 1–3 lateral marginal setae in both sexes, third and fourth tergites separately with 2 median discal and 1–3 pairs of lateral discal setae in both sexes…………11M. nudigena Mesnil-M. brunneisquama Zhang and Li sp. nov.Abdominal syntergite 1 + 2 without median marginal setae in both sexes.…………….…………..………………Abdominal syntergite 1 + 2 with two strong median marginal setae in male and weak setae in female. fourth tergite with 2–4 median discal setae………….Macquartia brunneisquama Zhang and Li sp. nov.Type material. HOLOTYPE ♂: China, Hubei, Yichang, Dalaoling National Natural Reserve, 1690–1790 m, 31°4′35.6″ N, 110°56′11.6″ E, 11–27 July 2017, malaise traps (BFU = Beijing Forestry University). PARATYPES. 5♂, same as holotype. 1♀, Qinghai, Qilian Mountains, Zhamasi, 3050 m, 38.90° N, 99.59° E, 19 August 2019, Li JJ .Diagnosis. Male frons as wide as anterior ocellus, parafacial hairy at most on upper half, ocelli yellow. Palpi reddish yellow. Two presutural and three postsutural dorsocentral setae. Legs black, mid tibia with two anterodorsal setae. Lower calyptrae divergent from scutellum. Middorsal excavation of abdominal syntergite 1 + 2 not extending to its posterior margin, with a pair of lateral marginal setae; third tergite with two median marginal and two pairs of lateral marginal setae, without median discal seta.Description. Body length 4.2–5.5 mm.Male. Head black, with sparsely grayish white pruinosity on fronto-orbital plate, inner parafacial and face, but dense on outer parafacial; lower face, lower occiput and genal dilation with dark grayish pruinosity; frontal vitta dark brown; ocelli whitish yellow; lunule dark brown; upper occiput black. Pedicel dark brown to reddish brown; the basal half of first flagellomere reddish yellow, apical half of first flagellomere dark brown; palpus reddish yellow. Frons strongly narrowed above, at the narrowest point 1–1.2 times as wide as anterior ocellus, in profile 1.6–1.8 times as long as the face; frontal vitta almost linear in front of ocellar triangle and strongly widened anteriorly; parafacial nearly parallel-side, 1.2–1.5 times as wide as (slightly narrower in profile) first flagellomere; face weakly concave, very weakly carinate on upper median portion between base of antenna, lower portion very weakly but warped forward; gena approximately 1/4 in profile as high as eye height; occiput flatted, not bulged. Inner vertical setae hair-like, not different from postocular setae row; ocellar seta slender, about 2/7 as long as eye height; 8–9 frontal setae, 2 upper setae finer and shorter, lowest seta nearly with middle level of pedicel; fronto-orbital plate with dense hairs, parafacial almost bare, only with 2–3 hairs below lowest fronto-orbital seta; 8–10 long and strong subvibrissal setae. Base of antenna nearly level with middle of eye; antenna short, at most 3/5 as long as face; pedicel with a long seta which is nearly as long as the first flagellomere; first flagellomere about 2.2 times as long as wide and 2 times as long as pedicel; arista short pubescent, about 1.5 times as long as pedicel and first flagellomere together, thickened on basal 1/5–1/6, second aristomere as long as wide. Prementum 2–2.5 times as long as wide, about as long as genal height; palpus about 1.3 times as long as first flagellomere.Thorax black in ground color, with very thin grayish pruinosity on postpronotal lobe, presutural area of scutum and proepimeron. Hairs black, rather sparse short and erect or suberect on scutum and scutellum, and dense on pleura; one 1 presutural and 1–2 postsutural acrostichal setae; 2 presutural and 3 postsutural dorsocentral setae; 2 postsutural intra-alar seta; prealar seta weak slender, as wide as the second supra-alar seta; 2 postpronotal setae; 2 pairs of reclinate and strong marginal scutellar setae; apical scutellar setae about twice as long as scutellum; a pair of discal scutellar setae, about as long as scutellum. Two katepisteral setae and anterior one weak; one upper anterior and a row of (4–6) posterior anepisternal setea; one weak anepisternal seta.4+5 with 2–3 fine setulae dorsally and ventrally. Relative lengths of costal sectors second, third, and fourth approximately as 1:4:1.3; vein M from dm-cu crossvein to its bend about 5.5 times the distance between the bend and wing hind margin.Wing hyaline, weakly tinged with pale brown, more strongly tinged on basal portion; tegula and basicosta dark brown; lower calypter divergent from scutellum, brown to dark, expect the basal brownish; halters yellow. Costal spine short, approximately 0.7 times the length of crossvein r-m, base of vein RLegs black or dark brown, pulvilli yellowish, claws and pulvilli nearly as long as or longer than fifth tarsomere. Fore tibia with 2–3 shorter anteriodorsal setae, preapical anterodorsal seta about as long as preapical dorsal seta; mid tibia with 2 anterodorsal seta, upper one short, 2 posterior setae, 1strong ventral seta; hind tibia with a row of irregular anterodorsal, 2 of them strong, 2posterodorsal, and 2ventral setae, upper one short; 1 preapical anterodorsal seta shorter than 1preapical dorsal seta.Abdomen long ovate, without pruinosity. Middorsal excavation of syntergite 1 + 2 not extending to its posterior margin, with 1–2 lateral marginal seta, and with some lateral discal setae; third tergite with two strong median marginal setae and 1–3 lateral marginal setae, without discal seta; fourth tergite without discal seta; fifth tergite with a row of discal setae. Base of sternite five distinctly convex, the depth of V-shaped median cleft of sternite five short, about 2/9 of the sternite length, inner margin of posterior lobe slightly pointed at apex. Pregonite short and bluntly rounded, postgonite triangular, short and pointed at apex, basiphallus cylindric, distiphallus short triangular and membranous. In caudal view, cerci thick at basal 3/4 and apical 1/4 narrowed and point, surstylus very short and slightly bent outward. In a lateral view, cerci slender and its apex slightly bent backward, surstylus bluntly round at apex.Female. Differing from male as follows: frons wide, vertex about 1/3 of head width; frontal vitta at middle about twice as wide as fronto-orbital plate; 6–8 frontal setae, and with 2 proclinate orbical setae; 1 upward prevertical seta, about as long as occellar, but weaker than orbical seta; inner vertical setae strong, distinct different from postocular setae row; outer vertical setae about 3/4 as long as inner vertical setae; lunule brown; pedicel and the basal of first flagellomere reddish yellow. Claws and pulvilli shorter than fifth tarsomere.Etymology. The specific epithet is taken from a male character of this species, i.e., lower calypter brown. It is derived from the Latin adjective brunneus (=brown) and the Latin noun squama .Distribution. China .Remarks. The new taxon differs from other species of Macquartia in having a narrow male frons, as wide as anterior ocellus, parafacial usually bare, ocelli yellow. Palpi reddish yellow. Gena approximately 2/5 in profile as high as eye height; two presutural and three postsutural dorsocentral setae. Lower calyptrae yellowish, divergent from scutellum. Legs black, mid tibia with two anterodorsal setae. Middorsal excavation of abdominal syntergite 1 + 2 not extending to its posterior margin, third tergite with two median marginal and two pairs of lateral marginal setae, without median discal seta.Macquartiachinensis Zhang and Li sp. nov.Type material. HOLOTYPE ♂, China, Hubei, Yichang, Dalaoling National Natural Reserve, 1690–1790 m, 31°4′35.6″ N, 110°56′11.6″ E, 11–27 July 2017, malaise traps (BFU). PARATYPES. 6♂2♀, same as holotype (BFU). 3♂, Liaoning, Qingyuan, Wandianzi, 600–720 m, 41.97° N, 115.29° E, 21 July 2016, Li XY, Liang HC. 1♀, Yunnan, Lijiang, Yulong Snow Mountains, High Mountain Botanical Garden, 2685–3107 m, 26.90° N, 100.17° E, 20 July 2017, Liang HC (SYNU).Diagnosis. Frons of male 1/3 as wide as first flagellomere. Parafacial bare. Pedicel reddish yellow, three presutural and three postsutural dorsocentral setae. Lower calyptrae divergent from scutellum. Mid tibia with 1 anterodorsal seta. Middorsal excavation of syntergite 1 + 2 not extending to its posterior margin, without lateral marginal seta, third and fourth tergites without median discal seta.Description. Body length 4.9–7.1 mm.Male. Head black, with sparsely grayish white pruinosity on fronto-orbital plate, parafacial and face; lower face, lower occiput, and genal dilation with dark grayish pruinosity; frontal vitta dark brown; ocelli reddish yellow to red; lunule dark brown to yellowish brown; upper occiput black. Antenna reddish yellow, except for apical half of first flagellomere dark brown; palpus reddish yellow. Frons strongly narrowed above, at the narrowest point as wide as base of arista or anterior ocelus, in a profile 1.4–1.6 times as long as the face; frontal vitta almost linear in front of ocellar triangle and strongly widened anteriorly; parafacial nearly parallel-side, about twice as wide as the first flagellomere; face weakly concave, lower portion very weakly but warped forward; gena about 2/9 (about 1/8–1/4 in profile) as high as eye height; occiput flatted, not bulged. Inner vertical setae fine and long, hair-like, not different from postocular setae row; ocellar seta slender, fine and long about 0.2–0.23 times as long as eye height; 9–10 frontal setae, 3–4 upper setae finer and shorter, lowest seta nearly with middle level of pedicel; fronto-orbital plate with hairs, parafacial almost bare, only with 3–6 hairs below lowest fronto-orbital seta; a row of (9–10) subvibrissal setae, which at most about 1/2 as long as vibrissal. Base of antenna nearly level with the middle of the eye; antenna short, 0.57–0.64 times as long as face; pedicel with a slender seta which is nearly as long as first flagellomere; first flagellomere 2.5–2.7 times as long as wide and two times as long as pedicel; arista short pubescent, about 1.5 times as long as pedicel and first flagellomere together, thickened on basal 1/5–1/6, second aristomere 1.2 times as long as wide. Prementum about 2.5 times as long as wide, about as long as genal height; palpus about 1.5 times as long as first flagellomere.Thorax black in ground color, with very thin grayish pruinosity on postpronotal lobe, presutural area of scutum and proepimeron. Hairs black, rather sparse short and erect or suberect on scutum and scutellum, and short on pleura; one presutural and one postsutural acrostichal setae; three presutural and three postsutural dorsocentral setae; two postsutural intra-alar seta; prealar seta about as long as notopleural, and slightly longer or as wide as second supra-alar seta; two postpronotal setae; two pairs of reclinate and strong marginal scutellar setae; apical scutellar setae crossed, about twice as long as scutellum; two discal scutellar setae. Two katepisteral setae and anterior one weak; one upper anterior and a row of (4–6) posterior anepisternal setea; 1 weak anepisternal setae; anatergite hairy.4+5 with 2–4 fine setulae dorsally and ventrally, Relative lengths of the costal sectors second, third, and fourth approximately as 1:3.5:1.2; vein M from dm-cu crossvein to its bend about 4.5 times distance between the bend and wing hind margin. The end of M and R4+5 nearly closed at wing margin.Wing hyaline, weakly tinged with pale brown, more strongly tinged on basal portion; tegula and basicosta dark brown; lower calypter pale brownish, fringe yellow; halters yellow. Costal spine short, about 1/2 length of crossvein r-m, base of vein RLegs black, pulvilli yellowish, claws and pulvilli slightly longer than fifth tarsomere. Fore tibia with 3 short anteriodorsal setae, upper 3–4 short and weak, preapical anterodorsal seta about as long as preapical dorsal seta; mid tibia with 1 anterodorsal seta, 2–3 posterior setae, 1 ventral seta; hind tibia with 2–3 anterodorsal, 2 posterodorsal and 2 ventral setae; 1 preapical anterodorsal seta shorter than 1 preapical dorsal seta.Abdomen long ovate, without pruinosity. Middorsal excavation of syntergite 1 + 2 not extending to its posterior margin, without median marginal and lateral marginal seta, but with some lateral discal setae; third tergite with 2 median marginal setae and 1–2 lateral marginal setae, without discal seta; fourth tergite without discal seta; fifth tergite with 4–6 discal setae. Sternite 5 nearly triangular, the depth of V-shaped median cleft of sternite 5 about 1/3 of the sternite length, posterior lobe bluntly rounded apically. Pregonite short, postgonite thin and pointed, distiphallus sclerotized, thin and long. In caudal view, cerci and surstylus short and slightly pointed at apex. In lateral view, cerci short, distinctly bent backward, surstylus thick and long, and slightly rounded at apex.Female. Differing from male as follows: frons wide, vertex about 1/3 of head width; frontal vitta at middle slightly wider than fronto-orbital plate; 5–6 frontal setae, and with 2 proclinate orbical setae; 1 outward prevertical seta, about as long as occellar, but weaker than orbical seta; inner vertical setae strong, outer vertical setae is 0.6–0.8 times as long as inner vertical setae; antenna reddish yellow, except for first flagellomere apex dark brown. Apical 2/3 of palpus usually inflated. Claws and pulvilli shorter than fifth tarsomere.Etymology. The specific epithet is taken from the locality of this species, i.e., China.Distribution. China .Remarks. This species is distinguished from the other species of this genus in having reddish yellow pedicel, three presutural and three postsutural dorsocentral setae, mid tibia with one anterodorsal setae; middorsal excavation of abdominal syntergite 1 + 2 not extending to its posterior margin, without lateral marginal setae, tergites 3 and 4 without median discal seta.Macquartia flavipedicel Zhang and Li sp. nov.Type material. HOLOTYPE ♂, China, Hubei, Yichang, Dalaoling National Natural Reserve, 1690–1790 m, 31°4′35.6″ N, 110°56′11.6″ E, 11–21 July 2018, malaise traps (BFU). PARATYPES. 18♂1♀, same as holotype (SYNU).Diagnosis. Parafacial bare, at most with 3–4 hairs below lowest frontal seta. Ocelli whitish yellow. Aristal hairs at least more than a diameter of the aristal base. Palpi reddish yellow. Thoracic scutum with gray pruinosity, three postsutural dorsocentral setae, two katepisternal setae. Lower calyptrae divergent from scutellum. Middorsal excavation of syntergite 1 + 2 extending or nearly to its posterior margin, with a pair of lateral marginal setae.Description. Body length 4.9–7.0 mm.Male. Head black, with sparsely grayish white pruinosity on fronto-orbital plate, parafacial and face; lower face, lower occiput and genal dilation with dark grayish pruinosity; frontal vitta dark brown; ocelli whitish yellow; lunule yellow; upper occiput black. Antenna reddish yellow, except for the apical half of first flagellomere dark brown; palpus reddish yellow. Frons strongly narrowed above, at the narrowest point 1/2–2/3 times as wide as antenna, in a profile 1.3 times as long as the face; frontal vitta almost linear in front of ocellar triangle and strongly widened anteriorly; parafacial nearly parallel-side, 2.0–3.0 times as wide as (nearly as wide as in profile) the first flagellomere; face weakly concave, lower portion very weakly but warped forward; gena about 1/5 (about 1/4 in profile) as high as eye height; occiput flatted, not bulged. Inner vertical setae fine and long, hair-like, not different from postocular setae row; ocellar seta slender, fine and long, about as long as 2/9 of the eye height; 8–10 frontal setae, 2 upper setae finer and shorter, lowest seta nearly with middle level of pedicel; fronto-orbital plate with dense hairs, parafacial almost bare, only with 3–4 hairs below lowest fronto-orbital seta; 5–7 long and strong subvibrissal setae; postocular setae close to posterior eye margin, long and directed forward on upper 1/2. Base of antenna nearly level with middle of eye; antenna short, at most 2/3 as long as face; pedicel with a long seta which is nearly as long as the first flagellomere; first flagellomere about twice as wide and 1.5 times as long as pedicel; arista short pubescent, about 1.5 times as long as pedicel and first flagellomere together, thickened on basal 1/5–1/6, second aristomere as long as wide. Prementum 2–2.5 times as long as wide, about as long as genal height; palpus about 1.5 times as long as first flagellomere.Thorax shining black in ground color, with very thin grayish pruinosity on postpronotal lobe, presutural area of scutum, and proepimeron. Hairs black, rather sparse short and erect or suberect on scutum and scutellum, and dense and short on pleura; 1 presutural and 1 postsutural acrostichal setae; 3 presutural and 3 postsutural dorsocentral setae; 2–3 postsutural intra-alar seta, if 3 setae present, anterior one weak; prealar seta weak or hair-like, distinctly shorter than first supra-alar seta; 2 pairs of reclinate and strong marginal scutellar setae; apical scutellar setae crossed, about twice as long as scutellum; a pair of discal scutellar setae near apex, about as long as scutellum. Two katepisteral setae and an anterior weak one; one upper anterior and a row of (4–6) posterior anepisternal setea; one weak anepisternal seta, thin and long.4+5 with 3–4 fine setulae dorsally and ventrally, relative lengths of costal sectors second, third, and fourth approximately as 1:2.6:1; vein M from dm-cu crossvein to its bend at approximately six times distance between the bend and wing hind margin.Wing hyaline, weakly tinged with pale brown, more strongly tinged on basal portion; tegula and basicosta dark brown; lower calypter pale brownish, fringe yellowish; halters yellow. Costal spine short, about 1/2 length of crossvein r-m, base of vein RLegs black or dark brown, pulvilli yellowish, claws and pulvilli nearly as long as or longer than the fifth tarsomere. Fore tibia with 2–5 anteriodorsal setae, upper 3–4 short and weak, preapical anterodorsal seta about as long as preapical dorsal seta, 1 posterodorsal; mid tibia with 1 anterodorsal seta, 2 posterior setae, 1 ventral seta; hind tibia with a row of irregular anterodorsal, 3 or 4 of them strong, 2 posterodorsal and 2–3 ventral setae; 1 preapical anterodorsal seta shorter than 1 preapical dorsal seta.Abdomen long ovate, without pruinosity. Middorsal excavation of syntergite 1 + 2 nearly extending to its posterior margin, without median marginal seta and lateral marginal seta, but with some lateral discal setae; third tergite with 2 strong median marginal setae and 1–2 lateral marginal setae, without discal seta; fourth tergite without discal seta; fifth tergite separately with a row of discal setae. Sternite 5 nearly rectangular, the depth of V-shaped median cleft short is about 1/6 of the sternite, inner margin of posterior lobe pointed at apex. Pregonite broad, postgonite short, apex bent downward and pointed hook-like, distiphallus thick and flat at apex. In caudal view, apical half of cerci slightly thick and apex pointed, surstylus short and bluntly round at apex. In lateral view, cerci slender and its apex slightly bent backward, surstylus short and thick, bluntly rounded at apex.Female. Differing from male as follows: frons wide, vertex 0.28–0.29 of head width; frontal vitta at middle slightly wider than fronto-orbital plate; 5–6 frontal setae, and with 2 proclinate orbical setae; 1 outward prevertical seta, about as long as occellar, but weaker than orbical seta; inner vertical setae strong, distinct different from postocular setae row; antenna reddish yellow, except for first flagellomere apex dark brown, claws and pulvilli shorter than fif5th tarsomere.Etymology. The specific epithet is taken from a character of this species, i.e., pedicel and basal half of flagellomere 1 reddish yellow. It is derived from the Latin adjective flavus (=yellow) and the Latin noun pedicul .Distribution. China (Hubei).Remarks. This new species resembles M. pubiceps but can be characterized by yellow pedicel and basal half of flagellomere, three postsutural dorsocentral setae, basicosta dark brown, legs black, third and fourth tergites without median discal seta in both sexes.Macquartia flavifemorata Zhang and Li sp. nov.Type material. HOLOTYPE ♀, China, Hubei, Yichang, Dalaoling National Natural Reserve, 1690–1790 m, 31°4′35.6″ N, 110°56′11.6″ E, 11–27 July 2017, malaise traps (BFU). PARATYPES. 1♂8♀, same as holotype (SYNU).Diagnosis. Parafacial bare. Pedicel and basal half of flagellomere yellow, palpi, basicosta, and legs reddish yellow except dark tarsi, aristal hairs at least more than diameter of aristal base. Apical half of scutellum brown to yellowish. Two presutural and three postsutural dorsocentral setae, two katepisternal setae. Lower calyptrae divergent from scutellum. Middorsal excavation of syntergite 1 + 2 nearly to 4/5 of its posterior margin, third tergite with two weak median marginal setae in male, third and fourth tergites without median discal setae in both sexes.Description. Resembles Macquartia flavipedicel sp. nov., but it is distinguished as follows:Body length 4.0–6.5 mm.Male. Frons strongly narrowed above, at the narrowest point 1/2–1/3 times as wide as antenna, in profile 1.2–1.5 times as long as face; parafacial about twice as wide as (about 1/2 as wide as in profile) first flagellomere; gena about 1/4 (about 1/4 in profile) as high as eye height; inner vertical setae fine and long, different from postocular setae row; ocellar seta slender, about 1/5 as long as eye height; first flagellomere about 2.5 times as long as wide and 2.2 times as long as pedicel. Thorax. With four dark longindinal thoracic vittae. Apical half of scutellum brown to yellowish. Two presutural and three postsutural dorsocentral setae; two postsutural intra-alar seta; without prealar seta; two supra-alar setae. Wing. Tegula dark brown; basicosta reddish yellow. Relative lengths of costal sectors second, third, and fourth approximately as 1:3.6:1.2; vein M from dm-cu crossvein to its bend about five times the distance between the bend and wing hind margin. Legs yellow, expect tarsi dark brown. Abdomen. Third tergite without median marginal seta; fifth tergite with two discal setae. Base of sternite 5 bluntly round, the depth of V-shaped median cleft of sternite 5 about 1/5 of the sternite length, posterior lobe bluntly rounded apical. Pregonite short and thick, postgonite short and pointed, distiphallus narrowed on apical half, and bluntly round at apex. In caudal view, cerci thick and short, apex bent backward, surstylus thick and apex slightly bent inward. In lateral view, cerci thick and its apex bent backward, surstylus thick and spoon-like, apex bent front-outward.Female. Differing from male as follows: frons wide, vertex 0.27–0.3 of head width. Parafacial bare. Frontal vitta wider than fronto-orbital plate, latter with sparse black hairs. Seven frontal setae, one outward prevertical seta, two proclinate outer orbital setae, outer vertical seta about 1/2 length of inner vertical seta. Tegula brown at base and reddish at apical half, basicosta reddish yellow. Pulvilli yellowish, claws and pulvilli shorter than fifth tarsomere. Fore tibia with 2–3 anterodorsal setae, lower one longer, 1 preapical anterodorsal and 1 preapical dorsal seta in equal length, and 1 preapical posteroventral seta. Mid femur with 2–3 anterior setae and 2 preapical posterodorsal setae. Mid tibia with one anterodorsal, two posterior, and one anteroventral seta, apex with one anterior, one anterodorsal, one dorsal, one posterodorsal, one anteroventral, and one posteroventral seta. Hind femur with a complete row of anterodorsal setae, one anterior and three ventral  setae. Hind tibia with a row of irregular anterodorsal setae, lower two of them strong, two posterodorsal and three anteroventral (lower two longer) setae, one preapical dorsal, one short anterodorsal and one preapical anteroventral seta. Abdomen without discal seta on intermediate tergites, syntergite 1 + 2 with 2–3 lateral marginal setae; third tergite with two median marginal setae or absent, with 2–3 lateral marginal setae; fifth tergite with a complete row of discal setae.Etymology. The specific epithet is taken from a character of this species, i.e., legs reddish yellow. It is derived from the Latin adjective flavus (=yellow) and the Latin noun femur (=femora).Distribution. China (Hubei).Remarks. This new species is similar to Macquartia flavipedicel sp. nov., but differs from it having reddish yellow basicosta and legs. The major differences of four new species are listed  and a control region, respectively . The gencquartia . The fouMacquartia species exhibit the same codon preference pattern  can directly indicate the preference of codon usage, and four  pattern . The aminad6 (Pi = 0.110), nad5 (Pi = 0.096), nad2 (Pi = 0.092), and atp8 (Pi = 0.084), while nad1 (Pi = 0.062), nad4L (Pi = 0.071), nad3 (Pi = 0.072), and cox1 (Pi = 0.076) displayed comparatively low Pi values, indicating that they are the most conserved genes among the 13 PCGs. The genetic distance and Ka/Ks analyses also verge on virtually identical , nad5 (0.104), and nad2 (0.1) have undergone a relatively fast evolution. Conversely, nad1 (0.066), nad4L (0.076), and nad3 (0.077) with lower distances are evolving comparative slowly. The genes with the lowest and highest Ka were cox1 (0.018) and nad6 (0.065), respectively, while the lowest and highest Ks genes were nad1 (0.172) and cytb (0.28), respectively  in the most clades .Janthinomyia sp. as a sister group to Phasiinae. The monophyly of the three remaining subfamilies are confirmed, with the exception of Dexiinae, which is represented by only one species. The Rutilia goerlingiana established a distinct clade at the base of the Tachinidae, while we only used one tribe in our phylogenetic studies and the number of taxa sampled was too little to be typical. As a result, further data of Dexiinae mitogenomes are needed to validate the monophyly of this subfamily. More tachinid sequences are also required to examine the connections between Dexiinae and other subfamilies. Stireman et al. disagree with the subfamily-level associations established herein using mitogenomic data, which used nuclear genes for phylogeny reconstruction [Palesisa + Clemelis) + Elodia + Compsilura) + Exorista) + (Winthemia + Nemorilla)). The included Macquartia species were shown to form a monophyletic clade, with the tribe Palpostomatini occupying a sister position to the Macquartiini; moreover, the PP value (0.95) is very high, which strongly supports this topology structure. Our results show that all members  are grouped into a clade with high support values (PP = 1). Within this group, M. brunneisquama, M. chinensis, and M. flavipedicel were clustered together and sisters to M. flavifemorata. We also found that M. brunneisquama and M. chinensis were placed into a subclade with strong nodal support values (PP = 1). As a consequence, this branch structure is still plausible and supports the conventional taxonomy. As only the mitogenomes of four species are detailed in this study, more species should be obtained to reveal the phylogenetic relationships of Tachinidae.The phylogenetic relationship of this study shows similarities compared with those based on the morphology and molecular data revealed in previous studies . The Tactruction . This intruction , Kutty etruction , and Yantruction . The triM. brunneisquama, M. chinensis, M. flavifemorata, and M. flavipedicel, were described and illustrated and their comparison with congeners as well as the identification keys from China were provided. Then, we newly sequenced four complete mitogenomes to describe the characteristics of the genomes and align the phylogenetic relationships with their related taxa. This is the first report of mitogenome sequences from the genus Macquartia of the tribe Macquartiini. After our comparisons to other complete mitogenomes of Tachinidae, the species of Macquartia were found to be consistent in gene order, gene location, codon usage, nucleotide composition, and AT-biased pattern, and were highly conserved. Although the control regions vary greatly in length, their structure has not changed much, which includes four basic conservative regions. Bayesian inference phylogenetic analyses based on PCGs produce well-resolved topologies with most branches having strong support. These results offer a valuable framework for Tachinidae and could reinforce our understanding of the taxonomic status and phylogenetic relationships of Tachinidae. Additional taxon sampling will be necessary to verify the monophyly of Tachinidae and elucidate the relationships between Tachininae and other subfamilies and define the internal structure of this group.In this study, the combination of morphology and mitogenomes has allowed us to discover four new species of Tachinidae and infer its phylogenetic relationship. The four new species of


Older adults’ daily conversations with other older adults enable them to connect to their surrounding communities and improve their friendships. However, typical aging processes and fluctuations in family caring might cause conversation changes. The purpose of this study was to explore the quantitative contributions of conversation turns (CTs) and speaking roles (SRs) in Mandarin-Chinese-speaking conversation dyads between mutually familiar healthy older adults (HOAs). A total of 20 HOAs aged 65 or over were recruited. Each dyad conversed for ten minutes once a week for five weeks, five sessions per dyad, for a total of 50 sessions. The frequency and percentages of the coded CTs and SRs contributed by each HOA were individually tallied and calculated. Quantitatively symmetrical contributions of CTs and SRs occurred in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. Although typical aging processes might change conversations, both Mandarin-Chinese-speaking HOAs serve as active interlocutors to each other in taking CTs and SRs to co-construct their conversation processes and content in their dyadic conversation. Sufficient knowledge of conversation co-constructions might lead them to have more supportive environments to connect to surrounding communities and improve their friendships. Senior Citizens Welfare Act, released in 2015 by the Ministry of Health and Welfare [In Taiwan, the  Welfare . Moreove Welfare , and mai Welfare . This is Welfare . In resp Welfare ,8. The n Welfare . Daily c Welfare ,12,13,14 Welfare ,17 can bHowever, typical aging processes  and fluctuating external environments  might cause conversation changes ,18,19. SA dyadic conversation is defined as two interlocutors participating in a conversation and involves collaboration influenced by a speaking interlocutor, a listening interlocutor, and conversation behaviors  ,25,26,27CTs are dominant elements of conversation across all modes of conversation behaviors   and critIn 1981, Goffman  proposedExtract 1A: 你有跟家人住嗎 B: 有和太太 A: 喔跟太太哦 Conversation content is used to determine the contributions of these three roles  from the interlocutors ,43,44,45Approximately 66.3% of Taiwanese people over six years of age speak Mandarin Chinese , and ManIn the past two decades, different perspectives of Mandarin-Chinese-speaking dyadic conversations were investigated, including cooperative and intrusive interruptions of Mandarin-Chinese-speaking and English-speaking healthy young adults  and discIn contrast, an intrusive interruption is a process wherein a listening interlocutor who disagrees with the speaking interlocutor’s messages intrusively interrupts to take the speaking interlocutor’s CTs  to disrupt the conversation process and/or content . These iLai  investigThe early studies in Mandarin-Chinese-speaking interlocutors reported two findings. First, while listening, Mandarin-Chinese-speaking healthy young adults cooperatively and intrusively consider the speaking interlocutors’ CT  interruptions in the simulations of doctor–patient interviews . HoweverAlthough quick alternations of CTs requiring substantial auditory attention-switching processes might be challenging for HOAs with typical aging processes  in dyadic conversations , Carter The current study was part of a large research project to explore the patterns of Mandarin-Chinese-speaking HOAs’ conversations. The study aimed to explore the quantitative contributions of CTs and SRs in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. Two research questions were investigated. First, do quantitatively symmetrical contributions of CTs occur in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs? Second, do quantitatively symmetrical contributions of SRs occur in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs? In view of these points, this study hypothesizes that the contributions of CTs and SRs in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs will be symmetrical.It has been emphasized that analyzing dyadic conversation is a suitable way to explore aging changes in Mandarin-Chinese-speaking conversation due to its single focus on a minimal set of a speaking interlocutor, a listening interlocutor, and simple alternations of CTs ,20. UndeTen dyads (D1 to D10) participated in the study, and each dyad contained two mutually familiar interlocutors, a total of 20 HOAs (ten men and ten women). The first ten HOA men  were recruited from local community groups and were requested to recommend one HOA woman  from their family and community. They all met the inclusion criteria of (a) being aged 65 or over; (b) using Mandarin Chinese or Taiwanese in their daily conversation environments; (c) having scores of Mini-Mental State Examination (MMSE) over 24 if educated or over 15 if uneducated; (d) hearing between 41–55 dB or better with naked ears or with hearing aids; and (e) being able to participate in basic social conversations. Although the occupation , gender , and attributes  of the HOAs might affect their language use , these dA quantitative, non-experimental, descriptive observation was conducted , and theThe occurrence of transcriptions  were only examined for reliability, and the exact duration of the pause time within and between the interlocutors was not examined due to not being the primary focus and not potentially affecting the current study. Competence was demonstrated when a minimum of 80% reliability on the transcriptions was reached for at least three of the segmented dyadic conversation sessions ,64,66.The coding training sessions first discussed the definitions of CTs and SRs. Second, these four research assistants independently coded two 5 min transcripts not collected in the current study and compared their coding with the pre-coded transcripts. The author and the research assistants discussed any discrepancies. Third, the research assistants independently coded CTs and SRs on two randomly selected 5 min portions of conversation transcripts, and these coded transcripts were compared to the pre-coded transcripts . Reliabihttps://www.datavideo.com/ (accessed on 1 December 2022), BC-50 1080p HD Block Camera) were located behind these two HOAs, and each video camera captured a frontal view of the head and mid-torso separately. These two captured views were inputted into a special effects generator  to video-record a split-screen view, in which the HOA was on one side of the split screen and the other HOA on the other side (see https://www.sony.com.tw/zh (accessed on 1 December 2022), UWP-D21), and the microphones were clipped to the HOAs’ clothing at approximately the sternum level, and the audio recordings were sent to a sound mixer , MG10XU MIXER). The video cameras recorded a split-screen view, which was played back on the 17-inch color monitor , and the audio recordings were monitored through a pair of audio headsets , ATH-M30x). The integration of video recordings and audio recordings was then stored on a secure digital memory card , Extreme SDXCⅠC10 U3 V30 64GB). The transcriptions were coded with CTs and SRs through Delve in New York, NY, USA (a registered trademark company: https://delvetool.com/ (accessed on 1 December 2022)), a software tool for analyzing qualitative data.Each core HOA casually conversed with their recommended HOAs in a quiet room where their daily conversation occurred, and these two HOAs were seated next to each other . Two colside see . The audEach dyad conversed once a week for five weeks, for a total of five dyadic conversation sessions per dyad and 50 dyadic conversation sessions. Sampling a greater amount of dyadic conversation sessions is needed to generalize validity . Each dyYour task today is to have a conversation with each other. Both of you will choose conversational topics. You might like to talk about daily living, including personal care, mobility, housework, and activities. No structured scripts will be used to keep the conversation as natural as possible. You will have about 10 min. I will signal you when 10 min has passed, so you may begin to wind up your conversation. If you feel you have finished your conversation before the end of 10 min, just let me know, and I will provide you with a list of possible topics to continue.The mean length of each dyadic conversation was 10 min 7 s, ranging from 10 min 0 s to 11 min 0 s.The data analysis involved three steps: (a) transcribing dyadic conversation sessions; (b) coding CTs; and (c) coding SRs  . The assSecond, CTs were independently coded on an utterance-by-utterance basis by R1 and R2 . A CT-A Third, three SRs  were independently coded on an utterance-by-utterance basis using Goffman’s  frameworAll 50 transcripts of the video-recorded conversation sessions from the ten dyads were coded by R1 and R2, with 25 transcripts for each. One transcript from each dyad, with ten transcripts in total, was randomly selected for calculating inter- and intra-coder reliability . R1 and t-test was conducted to compare the mean percentages of contributed CTs and SRs among these conversation dyads, similar to previous studies  contributed by each HOA in each dyad and the number of each animator, author, and principal were tallied. Percentages of the coded CTs and SRs contributed by each HOA in each dyad were calculated to account for the diverse length of the dyadic conversation sessions . The pers e.g., ,56,57).,57.t-test-test was calculated to compare the mean percentages of the CTs contributed by the core HOAs to the recommended HOAs among these 50 dyadic conversation sessions. The mean percentage of the core HOAs was 50.86 (sd = 6.40), and the mean percentage of the recommended HOAs was 49.14 (sd = 6.40). No significant difference  between the core and recommended HOAs was found (t(49) = 0.948, p > 0.05).The mean percentages of the contributed CTs for each HOA in the dyads are documented in t-test was calculated to compare the mean percentages of the SRs contributed by the core HOAs to the recommended HOAs. The mean percentage of the core HOAs was 51.08 (sd = 6.95), and the mean percentage of the recommended HOAs was 48.92 (sd = 6.95). No significant difference  between the core and recommended HOAs was found (t(49) = 1.099, p > 0.05).The mean percentages of the contributed SRs for each HOA in the dyads are documented in This study aimed to explore the quantitative contributions of CTs and SRs in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. Although typical aging processes might cause HOAs to have disturbances in speech , have declined language skills , and struggle to maintain conversations ,20, the The study revealed that quantitatively symmetrical contributions of CTs occurred in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. The two HOAs sequentially took their obligatory CTs to co-construct their conversation process in their dyadic conversation, as Extract 2 illustrates below. From line 1 to 6, these two HOAs  had orderly and equal CTs; three CTs contributed by A and B each. Accordingly, quantitatively symmetrical contributions of CTs can be anticipated and demonstrate the occurrence of an ideal conversation, as proposed by Sacks, Schegloff, and Jefferson . This maExtract 2A: 沒有辦法咬 B: 沒辦法咬就吃不下 A: 用果汁機也不好吃 B: 用果汁機可以吧 A: 那個營養差很多 B: 喔營養差很多 The study revealed that quantitatively symmetrical contributions of SRs occurred in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. These mutually familiar HOAs who shared personal experience and world knowledge took different SRs to co-construct conversation content  ,23. ExtrFurthermore, several factors could explain these symmetrical contributions of SRs. First, although the current study did not investigate the frequencies of cooperative and intrusive interruptions, the other-oriented  sensitivity in Mandarin Chinese proposed by Li (2001) might influence the HOAs to cooperatively interrupt the other HOAs to continuously co-construct their conversation content as illustrated in line 8 in Extract 3. Second, the HOAs did have the other HOAs act as the author and principal but had themselves act as an animator when co-constructing their conversation content. The other-oriented sensitivity in Mandarin Chinese proposed by Li (2001) might again influence the HOAs to use discourse-building features , as reported by Lai , to alteExtract 3B: 那女生她現在讀 A: 高二了 B: 高中二年級讀哪裡 A: 讀台中商職 B: 台中家商 A: 台中家商 B: 黑阿 B: (5.0) 坐公車去嗎 A: 不一定 B: 不一定 This study’s results should be interpreted with respect to three research limitations, which provide future research directions. First, although these HOAs all met the recruiting criteria, the validity of the findings might be influenced by diverse attributes  of the heterogeneous HOAs in Taiwan . The effThere are several practical implications from the findings of the current study. First, normative information on the quantitative contributions of CTs and SRs in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs can be acknowledged by the community care centers supervised by the MOHW  in TaiwaThis study explored the quantitative contributions of CTs and SRs in Mandarin-Chinese-speaking conversation dyads between mutually familiar HOAs. Quantitatively symmetrical contributions of CTs and SRs occurred in these Mandarin-Chinese-speaking dyadic conversations. Modesty, proposed by Wu , and oth


The existence of sex differences in disease incidence is attributed, in part, to sex differences in metabolism. Uncovering the precise mechanism driving these differences is an extraordinarily complex process influenced by genetics, endogenous hormones, sex-specific lifetime events, individual differences and external environmental/social factors. In fact, such differences may be subtle, but across a life span, increase susceptibility to a pathology. Whilst research persists in the hope of discovering an elegant biological mechanism to underpin sex differences in disease, here, we show, for the first time, that such a mechanism may be subtle in nature but influenced by multiple sex-specific factors. A proteomic dataset was generated from a gonadectomized mouse model treated with Tibolone, a menopausal hormone therapy. Following functional enrichment analysis, we identified that Alzheimer’s disease and the electron transport chain-associated pathways were regulated by sex-hormone interactions. Specifically, we identified that the expression of three respirasome proteins, NDUFA2, NDUFA7 and UQCR10, is significantly altered by compounding factors that contribute to sex differences. These proteins function in bioenergetics and produce reactive oxygen species, which are each dysregulated in many diseases with sex differences in incidence. We show sex-specific reprogrammed responses to Tibolone following gonadectomy, which primarily influence the expression of proteins contributing to metabolic pathways. This further infers that metabolic differences may underpin the observed sex differences in disease, but also that hormone therapy research now has potential in exploring sex-specific interventions to produce an effective method of prevention or treatment. Criteria for clinical trials did not include females as necessary subjects until the 1993 [Hormonal deprivation, akin to menopause and late-life testosterone decline, is a risk factor for many disorders. For example, Alzheimer’s disease affects twice as many women as men, and menopause is a known risk factor  linked tA potential mediator of these sex differences, and the relationship with hormonal deprivation, is the regulation of mitochondrial function by sex hormones, namely oestrogen, progesterone and androgens. This is evident in Alzheimer’s disease and Parkinson’s disease , which Of the many mitochondrial functions we observe as dysregulated, the electron transport chain is in focus here. It constitutes a series of protein complexes which may form sub-complexes or a supercomplex called the respirasome on the inner mitochondrial membrane. Together, the respirasome takes the products of glycolysis and of the Krebs cycle, passing electrons along each protein complex, driving protons out of the mitochondrial matrix and creating the mitochondrial membrane potential. This is essential to the production of ATP in this pathway. This process is critical to cellular productivity and homeostasis as the key producer of ATP, and its management of the damaging waste it produces, such as the production of reactive oxygen species by complex 1 and 3, is of interest. Its dysregulation can lead to an imbalance in energy production and increased cytotoxic conditions, potentially creating conditions more susceptible to pathologies .The dysregulation of mitochondrial function following hormonal deprivation is associated with increased risk of neurodegenerative diseases ,11. HoweTo explore these interactions, a male and female gonadectomised  mouse model was treated with Tibolone. This model can differentiate sex differences which are regulated by adult hormonal influence or genetics and/or postnatal hormonal organisation . Whole-bp < 0.15 and a protein–protein interaction analysis (PPI) followed by functional enrichment were assessed to identify those being regulated by all variables and significant associated clusters.To study the impact of key factors in sex differences (genetic/early life organisation and sex hormones) a proteomic dataset was generated from the prefrontal cortex in a gonadectomised model of male and female mice treated with Tibolone. Three samples from each test group underwent protein extraction followed by liquid chromatography with triple-TOF mass spectroscopy. This was then analysed using MaxQuant (v1.10.43) utilizing the integrated Andromeda search engine against the murine Uniprot database. This resulted in 2817 proteins with at least one identified peptide. This dataset was filtered to include two or more valid values per test group, resulting in 1135 proteins. These data were then transformed and missing values were imputed using Perseus. Following this, we performed a three-way ANOVA to identify proteins with significant changes in concentration following the impact of sex  and female (XX), each with their sex-specific early-life hormonal programming and puberty), gonadectomy , Tibolone (mice that received a Tibolone injection 24 h before sacrifice), and interactions between each factor . The prop < 0.15) in males (10p > −9.6), neuron adhesion (log10p > −9.9), Golgi-endoplasmic reticulum transport (log10p > −5.8), mitochondrial metabolism (log10p > −11), vesicle trafficking (log10p > −7.1) and platelet function (log10p > −6.8) (10p = −10.3), the establishment of organelle localisation (log10p = −8.9) and supramolecular fibre organisation (log10p = −8.7) was significantly enriched  . When thenriched . Utilisienriched .p < 0.15) in mice with gonads (10p > −12.5), mitochondrial metabolism (log10p > −8.5), the cytoskeleton (log10p > −6.9), endocytosis (log10p > −14.2), translation (log10p > −13) and vesicle trafficking (log10p > −4.8) (10p = −10.3), protein-containing complex assembly (log10p = −10.2) and carbon metabolism (log10p = −9.8) to be significant  . PPI ananificant e. Mappinnificant .p < 0.15. Of these, 106 proteins were upregulated (10p > −6.8), the citric acid cycle (log10p > −9.3), translation (log10p > −22.1), mitochondrial metabolism (log10p > −6.3), mRNA splicing (log10p > −13.7), endocytosis (log10p > −9.6) and statin pathways (log10p > −9.2) as being significant (10p = −11.9) and Alzheimer’s disease (log10p = −11.3)  e. Enrich= −11.3) .p < 0.15). A total of 119 proteins were identified to have their expression impacted by the interaction of sex and gonads, irrespective of Tibolone status (10p > −8.4), the electron transport chain (log10p > −16.5), ribosomes (log10p > −13.2), transcription (log10p > −12.2), prion diseases (log10p > −7.8), translation (log10p > −8), long-term potentiation (log10p > −10.8), oxidative phosphorylation (log10p > −8.8), proteosomes (log10p > −8), mRNA splicing (log10p > −6.2) and post-translation modifications (log10p > −6.8) to be significant  (10p > −6.4), translation (log10p > −12.6), respiratory complex 1 (log10p > −16.6), long-term depression (log10p > −9) and proteosomes (log10p > −7.9)  a. PPI an > −7.9) , with th > −7.9) .p < 0.15 to have gonad dependent responses to Tibolone in male and female mice (10p > −8.6), translation (log10p > −12.5), RAB signalling (log10p > −15.5), endocytosis (log10p > −10.2), the electron transport chain (log10p > −12.5) and the cytoskeleton (log10p > −7.8)  , and the > −7.8) .p < 0.15) (10p > −8.4), the electron transport chain (log10p > −16.5), translation (log10p > −13.2), WNT signalling (log10p > −12.2) and prion disease (log10p > −7.8)  . PPI ana > −7.8) . The enr > −7.8) .A meta-analysis of the proteins impacted by sex and hormone effects (gonadectomy and Tibolone), and interactions on protein expression was performed utilizing Metascape, an online pipeline for analysing multiple profiles a. We ide10p = −26.31, which, following multiple test adjustments was corrected to log10q = −22.3. The second most enriched function in this cluster, and overall, was ‘the citric acid cycle and respiratory electron transport’  from the pathway included across the lists. This pathway was determined to be highly enriched, with logansport’ . This re10p > −13.1), the RAF/MAP kinase cascade (log10p > −7.2), translation (log10p > −22.3), muscle contraction (log10p > −4.1), the citric acid cycle (log10p > −6.1), the dicarboxylic acid metabolic process (log10p > −7.1) and the cytoskeleton (log10p > −4.4) (10p > −13.3) and Alzheimer’s disease (log10p > −12.6) to be significantly enriched  . PPI anaenriched .p < 0.05 for sex–gonad–Tibolone interactions. The proteins identified were NDUFA2 (p = 0.042), NDUFA7 (p = 0.0047) and UQCR10 (p = 0.043)  c. NDUFA2Sex differences in neurological health and disease are underpinned, in part, by a number of biological factors which are genetic, hormonal or temporal in affect. The complexity of these compounding factors is substantial; as such, to understand diseases with sex inequality in incidence, further efforts must be made. This pressure is due to a lack of foresight in prior research design, as studies on sex differences in biological science may need to reconsider their design, and certainly adjust their analyses ,20. PresTo untangle some of these differences, we ran a proteomic analysis of the prefrontal cortex of an adult  gonadectomized  mouse model with a hormonal supplement (Tibolone). This allows us to interpret if changes in protein concentration between the sexes are regulated by gonadal hormones in adulthood or if such differences are influenced by sex-specific genetics/early-life hormonal organisation, and if there is potential to attenuate unwanted changes with hormonal supplementation . Our anap < 0.05) affected by sex–gonad–Tibolone interactions, as well as being present in a shared additional protein list of these top two enriched functions. Together, we believe this bolsters their potential as significant contributors to sex differences and their potential as novel sex-specific regulators of function.This analysis revealed the most significant cluster of enriched processes to contain many pathways associated with mitochondrial metabolism. The most enriched process overall was ‘Alzheimer’s disease’, which was followed by ‘the citric acid (TCA) cycle and respiratory electron transport’. Specifically, 24 respirasome proteins, whose expression was impacted by sex-associated factors and their interactions, were identified as shared between these two enriched processes. Of these proteins, the expression of three, NDUFA2, NDUFA7 and UQCR10, was identified to belong to the list of proteins significantly  may lack the statistical power to detect all of the sex differences in proteins contributing to pathways of interest which may create subtle cellular environmental changes, which may alter our susceptibility to a pathology across our lifetimes. However, this work has uncovered novel trends in sex-hormone interactions, such as those implied in our PPI and functional enrichment analyses, which can be used to inform the development of hypotheses for future studies [It is important to acknowledge that in this study, we set the alpha value for the inclusion of proteins in the functional enrichment and PPI analysis at 0.15. Our sample size  were kept under a 12 h light:12 h dark schedule. Mouse handling was performed in line with the NIH ‘Care and Use of Laboratory Animals guidelines’, and the ‘Use of Animals in Neuroscience Research’ by the Society for Neuroscience, and following the European Union guidelines (Council Directive 86/609/EEC).n = 3.Ethical approval was provided by the Institutional Animal Use and Care Committee  and by the Consejería del Medio Ambiente y Territorio . For the purpose of the experiment, a high number of animals would be most statistically healthy; however, given the novelty of the investigation, we chose to reduce the number of animals whilst allowing for a greater alpha value. Bilateral gonadectomy under halothane anaesthesia was performed at 2 months, with sham surgery in controls. At 3 months, 24 h before sacrifice, animals received one subcutaneous (sc) injection with 250 µg Tibolone  or vehicle (corn oil). Animal sacrifice was via cervical dislocation, brains were removed and 1g of pre-frontal cortex samples were collected before being delivered to the Centro Nacional de Biotecnología  for proteome analysis. This resulted in 8 test groups, with g for 15 min. The resulting supernatant was collected and quantified using a PIERCE 660 nm protein concentration assay. Protein digestion using an S-Trap filter  was performed with slight modifications. Briefly, 20 µg of protein of each sample was diluted to 40 µL with 5% SDS. Samples were reduced and alkylated by adding 5 mM tris(2-carboxyethyl)phosphine and 10 mM chloroacetamide for 30 min at 60 °C. Then, 12% phosphoric acid and then seven volumes of binding buffer  were added to the sample . After mixing, the protein solution was loaded to the S-Trap filter in two consecutive steps, separated by 2 min of centrifugation at 3000× g. Then, the filter was washed 3 times with 150 μL of binding buffer. Finally, samples were digested at 37 °C with Pierce MS-grade trypsin  at an enzyme:substrate ratio of 1:20 (w/w) in 20 μL of a 100 mM TEAB solution. Samples were spun in the S-Trap before digestion. Flow-through was then reloaded to the top of the S-Trap column and allowed to digest in a wet chamber at 37 °C overnight. To elute peptides, two step-wise buffers were applied ((1) 40 μL of 25 mM TEAB and (2) 40 μL of 80% acetonitrile and 0.2% formic acid in H2O), separated by 2 min of centrifugation at 3000× g in each case. Eluted peptides were pooled and vacuum-centrifuged to dryness. Digested samples were cleaned up/desalted using StageTips with Empore 3M C18 disks  [All samples were lysed with 500 µL of lysis buffer (5% SDS + 125 mM TEAB + 5 mM TCEP + 10 mM CAA + protease/phosphatase inhibitors). This mixture was incubated at 60 °C for 30 min, sonicated, and then, centrifuged at 16,000× ® M-Class Peptide BEH C18 Column  (Waters). The trap column was a C18 Acclaim PepMapTM 100 (Thermo Scientific) , switched on-line using the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µL/min. The nano-pump provided a flow rate of 250 nl/min and was operated under gradient elution conditions. Peptides were separated using a 150 min gradient ranging from 2% to 90% mobile phase B . The injection volume was 5 µL.A 1 µg aliquot of each digested sample was subjected to 1D-nano LC ESI-MSMS analysis using a nano-liquid chromatography system  coupled to a high-speed Triple TOF 5600 mass spectrometer  with a Nanospray III source. The analytical column used was a silica-based reversed-phase Acquity UPLCm/z) of 350 and smaller than m/z 1250 with a charge state of 2–5 and an abundance threshold of more than 90 counts (cps). Former target ions were excluded for 15 s. IDA rolling collision energy (CE) parameter script was used for automatically controlling the CE.Data acquisition was performed using a Triple-TOF 5600 System . Data were acquired using ion spray voltage floating (ISVF) of 2300 V, curtain gas (CUR) of 35, interface heater temperature (IHT) of 150, ion source gas 1 (GS1) of 25 and declustering potential (DP) of 100 V. All data were acquired in information-dependent acquisition (IDA) mode using Analyst TF 1.7 software . For IDA parameters, 0.25 s MS survey scans in the mass range of 350–1250 Da were followed by 35 MS/MS scans of 100 ms in the mass range of 100–1800 . Switching criteria were set to ions greater than mass-to-charge ratio  for both identification (FDR < 0.01 at the peptide level) and quantification .Mass spectrometry data obtained were analysed using MaxQuant  softwareRaw data were processed using Perseus  and filtMus musculus, with a confidence cut off of 0.40 for protein network formation. This was followed by functional enrichment analysis including GO biological process, GO cellular components, GO molecular functions, KEGG pathways and reactome pathways. Redundant terms were removed with a 0.5 cutoff. Related pathways were mapped using EnrichmentMap [q value < 0.05 and an edge cutoff <0.25. Enriched functions were then clustered into curated groups [Significantly expressed protein lists in each group were imported into cystoscope   and BioGrid are used to identify physical interactions. The PPI networks generated include proteins with at least 1 interaction. A molecular complex detection (MCODE) algorithm then groups associated interactors that share functionality.Utilizing Metascape , we perfp value < 0.01, a minimum count of 3, and an enrichment factor >1.5 were then grouped into clusters. p values were calculated based on a hypergeometric distribution, and q values were calculated using the Benjamini–Hochberg procedure. Kappa scores were used as the similarity metric when performing hierarchical clustering on the enriched terms, and sub-trees with a similarity of >0.3 were considered a cluster. The most statistically significant term within a cluster was chosen to represent the cluster. A term may be found enriched in several individual protein lists, and the best p value among them was chosen as the final p value.We analysed the shared functionally enriched pathways between each protein list sourced from GO biological processes, the KEGG pathway, reactome gene sets, CORUM, TRRUST, PaGenBase, WikiPathways and the PANTHER Pathway. Terms with a p values from each of the 20 clusters, with the constraint that there were no more than 15 terms per cluster and no more than 250 terms in total. The network was visualized using Cytoscape, where each node represents an enriched term and is coloured first by its cluster-ID. This was performed within Metascape [The clusters that were found to be of interest were used to prioritize the proteins that fell into those clusters. A subset of enriched terms was then rendered as a network plot, where terms with a similarity >0.3 were connected. We selected terms with the best etascape .www.graphpad.com. Venn diagrams were generated using an online webtool developed by the Van de Peer lab .Volcano plots were produced in R using the ggplot package . Heat man = 3, we elected for p < 0.15 to identify proteins with sexually dimorphic expression which are likely to be impacted by factors of sex differences; once collected, we utilized a cutoff of p < 0.05 for protein–protein interactions (PPI) and q < 0.05 when identifying functionally enriched pathways impacted by sex-associated factors.Our aim in the proteomic profile of this model was to identify subtle but clinically relevant differences between male and female mice which may contribute to sex differences in incidence across a lifetime. Sex differences are generally subtle and require a large number of test subjects to uncover . This raWe identified that the expression of NDUFA2, NDUFA7 and UQCR10, which each appear to be vital for the efficiency of respirasome activity, has sex-specific reprogrammed responses to Tibolone dependent on gonadal hormone status. Together, this suggests that future studies on hormone replacement therapies must consider sex- and gonad-specific interactions on protein expression, particularly for studies related to mitochondrial metabolism. More studies are warranted to uncover a potential sex-specific mechanism of action behind the reprogramming of mitochondrial metabolism-associated protein expression due to hormonal deprivation, akin to menopause, via Tibolone drug repurposing. This study also supports further investigation into hormone-based precision medicine intervention for women against sex-discriminating diseases such as Alzheimer’s disease, as it is possible that protective action may be time-dependent. Given the significant risk of menopause contributing to increased incidence of many diseases in women, a better-timed hormonal intervention for the individual could, in part, ameliorate this difference.