Tumor necrosis factor receptor-associated protein 1 regulates hypoxia-induced apoptosis through a mitochondria-dependent pathway mediated by cytochrome c oxidase subunit Ⅱ

Background:Tumor necrosis factor receptor-associated protein 1(TRAP1)plays a protective effect in hypoxic cardiomyocytes,but the precise mechanisms are not well clarified.The study is aimed to identify the mechanism of TRAP1 on hypoxic damage in cardiomyocytes.Methods:In this study,the effects of TRAP1 and cytochrome c oxidase subunit Ⅱ(COXⅡ)on apoptosis in hypoxia-induced cardiomyocytes were explored using overexpression and knockdown methods separately.Results:Hypoxia induced cardiomyocyte apoptosis,and TRAP1 overexpression notably inhibited apoptosis induced by hypoxia.Conversely,TRAP1 silencing promoted apoptosis in hypoxic cardiomyocytes.Further investigation revealed that the proapoptotic effects caused by the silencing of TRAP1 were prevented by COXⅡ overexpression,whereas COXⅡ knockdown reduced the antiapoptotic function induced by TRAP1 overexpression.Additionally,changes in the release of cytochrome c from mitochondria into the cytosol and the caspase-3 activity in the cytoplasm,as well as reactive oxygen species production,were found to be correlated with the changes in apoptosis.Conclusions:The current study uncovered that TRAP1 regulates hypoxia-induced cardiomyocyte apoptosis through a mitochondria-dependent apoptotic pathway mediated by COXⅡ,in which reactive oxygen species presents as an important component.

Cytochrome c Oxidase Subunit 1-based Human RNA Quantification to Enhance mRNA Profiling in Forensic Biology

RNA analysis offers many potential applications in forensic science,and molecular identification of body fluids by analysis of cell‑specific RNA markers represents a new technique for use in forensic cases.However,due to the nature of forensic materials that often admixed with nonhuman cellular components,human‑specific RNA quantification is required for the forensic RNA assays.Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology.The quantitative assay is based on real‑time reverse transcription‑polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range.The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

Epiphytic zooplankton community profiles in a typical urban wetland as revealed by DNA metabarcoding

Zooplankton,a crucial component of urban wetland,are one of the effective bioindicators for monitoring the feeding stocks of organisms at higher trophic levels and assessing the ecological quality of ecosystems.However,information about the characteristics of epiphytic zooplankton community structure resulted from traditional methods is limited and hindered by the large amount of detritus and sludge attached to the macrophytes.We investigated the epiphytic zooplankton communities associated with macrophytes(Vallisneria,Nymphaea,and Thalia dealbata)in a subtropical wetland using as DNA markers of the 18 S rRNA gene and the mitochondrial cytochrome c oxidase subunit I(COI)gene.A total of 241 OTUs of zooplankton were obtained from COI amplicons,including 194 OTUs of Rotifera,22 of Cladocera,and 25 of Copepoda,while only 62 OTUs of zooplankton were obtained from 18 S rDNA amplicons including 34 OTUs of Rotifera and 28 of Copepoda.The zooplankton communities associated with the three macrophytes were similar,but they differed significantly from those in the open waters.However,there were no significant temporal differences among the zooplankton communities.Epiphytic zooplankton communities were dominated by littoral zooplankton such as Testudinella,Lecane,and Philodina.Microzooplankton,especially littoral species,utilize macrophytes as food sources and as refuges against predation.This further led to an increase inαandβdiversity of zooplankton communities in urban wetlands.Our result suggests that the joint use of multiple molecular markers could improve the taxonomic resolution and generate a comprehensive biodiversity profile of zooplankton.

First record of the little fire ant,Wasmannia auropunctata(Hymenoptera:Formicidae),in Chinese mainland

In January 2022,we received ant specimens collected from three field colonies from Shantou City,Guangdong Province,China.They were identified as the little fire ant,Wasmannia auropunctata,through morphological and molecular analyses.Wasmannia auropunctata is listed as one of the 100 most dangerous invasive species by the International Union for Conservation of Nature(IUCN)and has spread from its native range in South America to every continent except Antarctica.DNA analysis of mitochondrial cytochrome c oxidase subunit I(COI)in nine specimens of W.auropunctata found that they had a close genetic relationship with specimens from Argentina.This study represents the first formal record of the establishment of W.auropunctata outdoor in Chinese mainland.However,the invasion stage and occurrence degree of W.auropunctata in China are not clear to date.The implementation of quarantine measures,investigation of the occurrence and distribution,and development of monitoring and control strategies are needed to actively respond to the threat posed by this highly invasive ant.

DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.

Morphological and molecular characterization of the rice root-knot nematode, Meloidogyne graminicola, Golden and Birchfeild, 1965 occurring in Zhejiang, China

The rice root-knot nematode Meloidogyne graminicola is a severe pest of rice. In China, it was first reported from Hainan Province, and later from several other provinces. In the present study, a rice root-knot nematode population found from the rice cultivation areas of Zhejiang Province, China is characterized via molecular analysis using internal transcribed spacer(ITS) and cytochrome c oxidase subunit Ⅱ(coxⅡ)-16 S rRNA genes and scanning electron microscopy(SEM) observations of males and the second-stage juveniles. Morphometric data and molecular sequence comparisons for all M. graminicola populations occurring in China are also provided. The overall morphology of M. graminicola found in Zhejiang match well with the original description, though males have a slightly longer body and stylet, and a shorter tail, while the second-stage juvenile is also slightly longer than in the original description. This is the first report of M. graminicola from Zhejiang. Phylogenetic studies based on coxⅡ suggest that all the Chinese populations belong to Type B. This study expands knowledge of the increasing distribution and phylogenetic relationships of M. graminicola that occur in China.

DNA barcode assessment of Ceramiales(Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales （Rhodophyta） were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene （LSU）, universal plastid amplicon （UPA）, and partial mitochondrial cytochrome c oxidase subunit I gene （COl）. No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Syrnphyocladia latiuscula, and Neosiphoniajaponica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD （Barcode of Life Data） Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might he a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H.japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

The complete mitochondrial genome of Triuncina daii(Lepidoptera:Bombycidae) and its phylogenetic implications

The bombycid moth, Triuncina daii Xing Wang ＆ Zolotuhin, 2015, plays an important role for analyzing the phylogenetic relationships of the family Bombycidae （Lepidoptera： Bombycoidea）. Here we first describe the complete mitochondrial genome （mitogenome） of T. daii, which includes thirteen protein-coding genes （PCGs）, twenty-two transfer RNA genes, two ribosomal RNA genes and an A＋T-rich region, and we find the mitogenome is 15,482 bp in length （GenBank no. KY091643）. The genes order and orientation in the T. daii mitogenome are similar to other sequenced lepidopteran species. Except for cox1, all of the PCGs started with ATN. Twelve PCGs stopped at TAA except for cox1 which stops at a single T. Thirteen PCGs of available species are used to demonstrate the inner phylogenetic relationships of Bombycoidea. The bombycid species form a monophyletic clade with a bootstrap value of 100% and a posterior probability of 1.00.

Prevalence and genotype distribution of Enterobius vermicularis among kindergarteners in Shiraz and Khorramabad cities, Iran

Objective: To study the prevalence and genotype of Enterobius(E.)vermicularis from adhesive tape samples in the cities of Shiraz and Khorramabad, Iran. Methods: A total of 1 000 adhesive tape samples from kindergartens in Shiraz(500 samples) and Khorramabad(500 samples) were collected and tested using a microscope to find E. vermicularis egg/s. A questionnaire was filled out for each sample. In order to characterize the genotype of E. vermicularis, the PCR-sequencing method of the mitochondrial cytochrome C oxidase subunit 1(cox1) gene was used. Genomic DNA was extracted from the positive scotch tape samples of E. vermicularis. The cox1 gene was amplified by the polymerase chain reaction and sequenced. The sequence data were aligned using the BioEdit software and compared with the published sequences in GenBank. Phylogenetic analysis was performed using the maximum likelihood method. Results: The parasitological method showed that 15 out of the 500 samples from Shiraz(3.00%) and 12 out of the 500 samples from Khorramabad(2.40%) were infected with E. vermicularis eggs. BLAST analysis indicated that the sequenced isolates belonged to E. vermicularis genotype B while three different haplotypes were also identified. Conclusions: This is the first study on genotyping E. vermicularis in the cities of Shiraz and Khorramabad. Considering the public health importance of the disease, further studies are necessary to characterize the genotype of E. vermicularis in human populations from other regions of Iran.

Genetic variation and phylogenetic relationship of Hypoderaeum conoideum(Bloch, 1782) Dietz, 1909(Trematoda: Echinostomatidae) inferred from nuclear and mitochondrial DNA sequences

Objective:To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR,as well as their phylogenetic relationship with American and European isolates.Methods:The nucleotide sequences of their nuclear ribosomal DNA(ITS),mitochondrial cytochrome c oxidase subunit 1(CO1),and NADH dehydrogenase subunit 1(ND1)were used to analyze genetic diversity indices.Results:We found relatively high levels of nucleotide polymorphism in ND1(4.02%),whereas moderate and low levels were observed in CO1(2.11%)and ITS(0.96%),respectively.Based on these polymorphisms,the 20 ND1,12 CO1,and 18 ITS haplotypes were classified,and several common haplotypes were observed in all samples.At least three major lineages,namely American,European and Asian lineages,have been classified by phylogenetic analyses based on ND1 sequences.Conclusions:Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum.However,a combination of all loci for ND1,CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite.Thus,comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.

Genetic diversity and population structure of the sea star Asterias amurensis in the northern coast of China

The sea star Asterias amurensis is widely viewed as a severe“marine pest”because of its broad feeding habits.Over the past few decades,A.amurensis undergoes massive and sporadic population outbreaks worldwide,causing extensive economic and ecological losses to the local aquaculture industry and marine ecosystem.Understanding the genetic diversity and population structure of A.amurensis can provide vital information for resource management.By analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I(COI)gene and ten simple sequence repeat(SSR)microsatellites markers,the genetic diversity and population structure of A.amurensis of four populations along the northern coast of China was uncovered.A total of 36 haplotypes were identified,and a main haplotype was found in four populations.The Qingdao(QD)population displayed the highest genetic diversity among all the populations.The AMOVA and pairwise F_(st)showed that there was small but statistically significant population differentiation among the four populations,especially between QD and Weihai(WH).Moreover,the principal component analysis(PCA)and admixture analysis showed that several individuals in Yantai(YT)and Dalian(DL)had little genetic association with other individuals.Overall,this study provided useful information of the genetic diversity and population structure of A.amurensis and will contribute to the resource management of A.amurensis in China.

Genetic difference of Chinese horseshoe crab(Tachypleus tridentatus) in southeast coast of China based on mitochondrial COI gene analysis

Population genetic structure and historical demography of Chinese horseshoe crab (T.tridentatus)along southeast coast of China were inferred from the sequence data of mitochondrial cytochrome c oxidase subunit Ⅰ (COI) fragment.The sequence analysis for 964 bp COI fragment was conducted in 28 individuals collected from five localities:Ninghai in Zhejiang Province,Meizhou and Zhangpu in Fujian Province,Beihai of Guangxi Zhuang Autonomous Region and Danzhou of Hainan Province.Sequence variation was relatively low with a total of seven transitions observed.In all localities,Haplotype H3 was the dominant type observed among eight haplotypes defined previously,and was at the center of radiation in Median-Joining network.The prolonged star-like network suggests a signature of population expansions.The level of diversity was low in total,with haplotype diversity ( Hd) being equal to 0.765 and nucleotide diversity (π) being equal to 0.00118,respectively.The genetic structure analysis revealed the significant genetic difference between Ninghai and Danzhou populations.Both mismatch distribution analysis and Fu's Fs test provided consistent inference of historic population expansion.The low genetic diversity of horseshoe crab observed along China coast indicated that urgent measures should be taken to protect this rare marine animal.

Genetic Variability of the Mitochondrial DNA in Honeybees （Apis mellifera L.） from Benin

The aim of this study was to evaluate the genetic variability in bees Apis mellifera from Benin by using mitochondrial DNA （mtDNA） as a molecular marker in their cytochrome c oxidase subunit I and II （COI-COI1） intergenic region. A total of 304 bee colonies were sampled in 27 municipalities of the cashew growing area of Benin. These samples were analyzed by the cleaved amplified polymorphisms technique for determining the haplotypes of subspecies present in the sampled population. Eight PCR-RFLP profiles of African lineage A were then identified in the 304 samples of bees investigated. Forty-nine percent （49%） of the samples showed the profile of haplotype A1 （subspecies adansonii of Zambia）, 40% of haplotype A4 （subspecies scutellata of South Africa） and 3% of haplotype A 19 （subspecies adansonii of Guinea）. Five other haplotypes of the African branch （A） that had been described in a previous study were also identified： new 1 （2%）, new 2 （2%）, new 3 （1%）, new 4 （2%） and new 5 （1%）. This study showed that A. rnellifera from Benin belonged only to lineage A with the predominance of haplotypes AI and A4. This study will contribute to the development of coherent policies for conservation of local bees in Benin.

Availability of Myoglobin as a Molecular Marker for Phylogenetic Relationships of Fish

In order to elucidate the phylogenetic relationship of fish, DNA and deduced amino acid sequences of myoglobin （Mb） were used for the phylogenetic analyses based on different approaches, namely, maximum likelihood （ML）, neighbor joining （NJ）, unweighted pair group method with arithmetic means （UPGMA） and maximum parsimony （MP） methods in comparison with the conventional molecular markers, mitochondrial cytochrome b （cyt-b） and cytochrome c oxidase subunit I （COI）. The phylogenetic trees drawn based on Mb sequences were similar to those by the traditional classification based on the other molecular markers. The primary and secondary structures, as well as the modeled tertiary structures of Mbs were similar to each other, but were clearly distinguishable among those species. Such differences in structure would be associated with adaptation of Mb molecule to the physiological conditions of each species. These results suggest that Mb can be a molecular marker for the phylogenetic relationship of fish.

Multi-gene-based investigation on the molecular phylogeny of the hypotrichous family Strongylidiidae(Protista,Ciliophora),with notes on the ontogeny of a new genus and new species

Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups,leading to their systematics remaining unresolved.Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data.In this study,a new strongylidiid genus Heterouroleptus is established,mainly based on the novel mode of origin of the ventral cirral rows:left ventral cirral row(LVR)originates from frontal-ventral-transverse cirral anlagen(FVTA)Ⅲ(anterior portion),IV(middle portion),and V(rear portion);right ventral cirral row comes from the entire FVTA VI.A new species,Hetero-uroleptus weishanensis gen.nov.,sp.nov.,is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense.Eight new sequences and nuclear gene markers(single-gene and multi-gene)are provided to analyze the phylogenetic relationships of strongylidiids,with the COI gene utilized to uncover further genetic information at species level and below.The results reveal that:(1)Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia;(2)Heterouroleptus gen.nov.forms a clade that is sister to all the other strongylidiids;(3)Hemiamphisiella Foissner,1988 and Pseudouroleptus Hemberger,1985 should not be synonyms,and both genera should be subdivided due to their variable morphological characteristics;(4)LVR originating from three anlagen is a plesiomorphy of Strongylidiidae.The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus,but also helps to improve the diagnosis of the family Strongylidiidae.

A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.

Phenotypic plasticity can explain evolution of sympatric polymorphism in the hairy snail Trochulus hispidus （Linnaeus, 1758）

Morphological variation of snails from the genus Trochulus is so huge that their taxonomy is unclear. The greatest variability concerns forms hispidus and sericeus/plebeius, which are often considered as separate species. To evidence the species barriers, we carried out crossbreeding ex- periments between these two sympatric morphs. Moreover, we compared the shell morphology of laboratory-bred offspring with their wild parents to test if the variation can be explained by the phenotypic plasticity model. We found that the two Trochulus morphs show no reproductive bar- riers. The fecundity rates, the mean clutch size, and F~ viability observed for all crosses were not significantly different. In hybrid crosses （in F2 generation）, we also recorded reproduction compati- bility, similar fecundity, and hatching success as in their parents. Accordingly, phylogenetic ana- lyses revealed the significant grouping of sequences from these different morphs and supported no constrains in reproduction between them. Comparison of shell morphology between wild and laboratory samples showed that various characters appeared highly plastic. The average shell shape of the hispidus morph changed significantly from flat with wide umbilicus to elevated with narrower umbilicus such as in the sericeus/plebeius morph. All these findings indicate that the examined morphs do not represent separate biological species and the evolutionary process is not advanced enough to separate their genetic pool. Therefore, phenotypic plasticity has played a sig- nificant role in the evolution of Trochulus shell polymorphism. The two morphs can evolve inde- pendently in separate phylogenetic lineages under the influence of local environmental conditions.

Analysis of Diversity of CO I Gene and System Generation of Nine Species of Chitons

Cytochrome c oxidase subunit 1 （CO I ） genes from 9 chiton species in China＇s coast area were sequenced. A phylogeny tree about these gene sequences were reconstructed together with other 4 CO I gene sequences of chitons from Genbank. The affin- ity genetic relationship and the taxonomic status of molecular evolution for these 13 chiton species were analyzed. The results show that Liolophura japonica and Onithochiton hirasei belong to Chitonidae, .4canthochiton rubrolineatus and Acanthochiton dis- similis belong to Acanthochitonidae, and Placiphorella japonica and Mopalia retifera belong to the same family Mopliidae. How- ever, Lepidozona coreanica, Ischnochiton comptus, and lschno- chiton hakodadensis are not supposed to be referred to Ischnochi- tonidae according to the genetic distance analysis （L. coreanica, 1. comptus, and 1. hakodadensis are usually classed as Ischnochi- tonidae according to their morphological character）. Furthermore, 1. hakodadensis could not been classed as lschnochiton, and it is more likely to be treated as a close relative of Lepidozona.

Mitochondrial and Nuclear DNA-based Identification of Some Forensically Important Calliphoridae(Diptera)in Luoyang of China

Introduction:Calliphoridae plays a key role in forensic entomology research,which is one of the first insects to decompose animal carcasses.The mitochondrial cytochrome c oxidase subunit I and the ribosomal internal transcribed spacer 2(ITS2)are among the most widely used molecular markers for insect taxonomic characterization.Aim:The aim of the study was to test the suitability of two genetic markers based on conducting the molecular identification of six necrophagous Calliphorid flies.Materials and Methods:Fourteen Calliphoridae flies were collected and classified with traditional morphological characteristics.The DNA of flies was extracted and the fragments of COI and ITS2 were amplified and sequenced.All the sequences were aligned and analyzed by MEGA 7 software for NCBI BLAST,nucleotide composition,intra-and inter-specific divergence calculation,and phylogenetic tree inference successively.Results:The results indicated that COI and ITS2 genes were robust in the identification of Calliphoridae at the species level and ITS2 gene sequence possessed a strong resolution power as it showed higher variation values between Lucilia sericata and Lucilia cuprina,Calliphora vomitoria and Triceratopyga calliphoroides,C.vomitoria andAldrichina grahami,but inferior to COI fbrT.calliphoroides and A.grahami.Conclusions:Our results showed that combination of COI+ITS2 genes yields more accurate identification and diagnoses and better agreement with morphological data than the mitochondrial barcodes alone.As a supplementary method for morphological identification,we advocated for the combination of nuclear and mitochondrial gene approaches to address the taxonomy and phylogeny of forensic relevant flies,especially of closely related species and populations.