First metagenomic sequencing for the analysis of microbial community population of Melophagus ovinus and pupae in Xinjiang, China

First metagenomic sequencing for the analysis of microbial community population of Melophagus ovinus and pupae in Xinjiang, China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First metagenomic sequencing for the analysis of microbial community population of Melophagus ovinus and pupae in Xinjiang, China Kaijun Huang, Xing Zhang, Qian Feng, Lu Sun, Na Xiong, Xiaoqing Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3990667/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Frontiers in Veterinary Science → Version 1 posted You are reading this latest preprint version Abstract Background Melophagus ovinus , a parasite on the body surface of sheep, directly attacks the host through biting and sucking blood and may also transmit pathogens in the process. There are currently only a few studies on the microbial composition of M. ovinus , while there are no such studies on pupae. Methods M. ovinus adults and pupae were collected from four regions in Xinjiang, China. DNA was extracted from the samples, amplified, and sequenced using the Illumina Novaseq 6000 System; finally, the sequencing data were analyzed using molecular biology software. Results From all samples, a total of 32 phyla, comprising 372 genera and 1037 species, were detected. The highest microbial diversity was observed in Kuqa City (AT-2) and Qira County (AT-4). Pupae exhibited 40 unique microbial genera (AT-5) but did not have the highest microbial diversity. Conclusions Proteobacteria was the dominant phylum in all samples. The dominant genera included Bartonella , Wolbachia , Pseudomonas , and Arsenophonus . This is the first study to report most of the bacteria (e.g., Bartonella bovis and Arsenophonus nasoniae ), fungi (e.g., Saitoella complicata ), viruses (e.g., Orf virus and Wolbachia phage WO), and protozoa (e.g., Trypanosoma theileri and Trichomonas vaginalis ) in M. ovinus . Melophagus ovinus Pupae Metagenomics Sequencing Microbial population Xinjiang Figures Figure 1 Figure 2 Figure 3 Background Melophagus ovinus is a blood-sucking parasite that parasitizes primarily on the body surface of sheep. This ectoparasite lacks wings and has a surface covered with bristles, robust mouthparts, and three pairs of sharp grappling hooks. M. ovinus is a member of the Hippoboscidae (Diptera, Hippoboscoidea) [ 1 ]; it also parasitizes the body surfaces of mammals, including Tibetan antelope [ 2 ], goats [ 3 ], rabbits [ 1 ], donkeys [ 4 ], and so on. The life cycle of this parasite consists of three distinct stages: larva, pupa, and wingless adult. Shortly after birth, female M. ovinus mates and produces offspring, giving birth to 12–15 offspring in her lifetime [ 1 ]. The geographic distribution of M. ovinus is very wide and it has been reported in Europe (Slovakia [ 5 ], France [ 6 ], England [ 7 ], Hungary [ 8 ], Croatia [ 9 ], Czechia [ 10 ], Poland [ 11 ] and Russia [ 12 ]), Asia (Turkey [ 13 ] and China [ 14 ]), South America (Peru [ 4 ]), North America (USA [ 15 ]), Oceania (Australia [ 12 ] and New Zealand [ 16 ]) and Africa (Algeria [ 17 ] and Ethiopia [ 18 ]). The parasitization of sheep with large amounts of M. ovinus results in two serious consequences. First, by biting and sucking blood, it makes the skin of sheep itchy and painful, leading to skin damage, wool loss, agitation, anemia, and weight loss. More severe cases can be secondary to pathogenic microbial infections and cutaneous myiasis [ 1 ]. The infestation can lead to a loss in the quality and production from sheep products, such as skin, wool, meat, and milk, adversely affecting the economy of the fur and livestock industries. Massive infections of M. ovinus led to a loss of 1.6 million USD by two Ethiopian tanneries over two years [ 19 ]. More importantly, M. ovinus carries a variety of pathogenic microorganisms that infect the host in the process of sucking blood, such as viruses (e.g., Bluetongue virus [ 20 ], Border disease virus [ 22 ] and African swine fever virus [ 23 ]), Acinetobacter spp. [ 24 ] (e.g., Ac. lwoffii [ 25 ]), Anaplasma spp. (e.g., An. ovis , An. Phagocytophilum , and An. bovis [ 26 ]), Arsenophonus spp. [ 27 ] (e.g., Ar. melophagi [ 28 ]), Bartonella spp. [ 11 ] (e.g., Ba. schoenbuchensis [ 6 ], Ba. chomelii [ 27 ], and Ba. melophagi [ 4 ]), Borrelia spp. (e.g., Bo. garinii , Bo. spirochetes belonging to the Bo. valaisiana -related group [ 29 ], and Bo. burgdorferi sensu lato [ 11 ]), Coxiella spp. (e.g., Co. burnetii [ 30 ]), Rickettsia spp. [ 8 ] (e.g., Ri. raoultii, Ri. slovaca [ 31 ], Ri. melophagi [ 10 ], Ri. massiliae , and Candidatus Rickettsia barbariae [ 14 ]), Theileria spp. (e.g., Th. ovis [ 2 ] and Th. Luwenshuni [ 32 ]), Trypanosoma spp. [ 11 ] (e.g., Tr. theodori [ 1 ] and Tr. melophagium [ 9 ]), Wolbachia sp. [ 24 ], etc. Most studies on M. ovinus have focused specifically on one to three pathogens. Concurrently, few studies have also detected and identified the types of viruses they carry or analyzed their microbial communities. Microorganisms colonize the body surface, intestine, hemocoel, and even cells of all insects, that serve as vectors for microorganisms. Their microbial composition and abundance can be altered by a variety of conditions, including insect species, sex, developmental stage, blood-sucking behavior, survival strategies, and geographic location [ 33 ]. Among these conditions, feeding on the blood reduces the bacterial diversity of the insect gut [ 34 , 35 ]. Insects and the microorganisms they carry may be in mutually beneficial symbiotic, parasitic, and competitive relationships. For example, microorganisms promote insect health, synthesize toxins or modify the insect immune system to protect it from pathogens, parasitize insects, and attack other hosts at opportune times [ 36 ]. The most important function of microorganisms in symbiotic association with insects is the provision of nutrients, digestion and detoxification [ 37 ]. The study of the microbial community composition and abundance of M. ovinus contributes to scientific knowledge concerning its biology and epidemiology. Metagenomic sequencing involves the high-throughput sequencing of the genomes of microbial communities in a sample, and facilitates the study of microbial population structure. This technique is free from the limitations of microbial isolation and pure culture, and can detect microorganisms in trace numbers, providing an effective tool for studying microbial communities in samples. Currently, only four studies have reported using high-throughput sequencing to analyze the microbial composition and diversity in M. ovinus . In 2017, Duan et al. [ 24 ] researched the characterization of midgut microbial populations in male and female M. ovinus , and in 2020, their team [ 27 ] extracted DNA from fully satiated, newly hatched and unfed female M. ovinus the midgut and whole body for bacterial community studies. Later in 2021, Litov et al. studied the virome of M. ovinus and annotated the full genomes of five novel viruses, and also proved that the Aksy-Durug Melophagus sigma virus can replicate in mammalian cells [ 12 ]. In 2022, Liu et al. [ 23 ] studied adult M. ovinus from three regions of Tibet and discovered for the first time, 23 bacterial genera and multiple DNA viruses. In 2023, Zhang et al. [ 39 ] sequenced, assembled, and annotated the full genome of M. ovinus . They also discovered that contractions and losses of sensory receptors and vision-associated Rhodopsin genes were significant in M. ovinus , and could contribute to the removal of host-sequence contamination during the high-throughput sequencing data processing. In this study, we employed the Illumina Novaseq 6000 System to metagenomic sequencing and analysis of the microbial composition and abundance in adult and pupae of M. ovinus from four regions in Xinjiang, China. This is the first report of the use of a technique has been used to study M. ovinus in the area surrounding the Taklamakan Desert, the second most mobile desert in the world, as well as the first study of microbial diversity in pupae. The blood-feeding behavior of insects significantly affects microbial composition and abundance within and outside their body. Thus, it is of great interest to study and analyze the microbial diversity of adult M. ovinus and pupae simultaneously from different regions. This study aims to provide scientific data for the discovery of new pathogens, assessment of their harm to public health and industrial economy, formulation of measures to prevent and control arboviruses spread, and the development of biological control methods. Methods Sample collection and DNA extraction M. ovinus adults and pupae (Fig. 1 ) were collected from four locations in Xinjiang, China, in 2019 to 2023 (Table 1 ). All hosts were sheep. M. ovinus was collected during the treatment of sheep for ectoparasites. The samples were stored in a -80℃ refrigerator until use. M. ovinus and its pupae were identified using morphological and molecular biology methods. For better description, the samples will be hence referred to by the abbreviations AT-1, AT-2, AT-3, AT-4, and AT-5. Table 1 Detailed sample information for Melophagus ovinus and pupae Location Sample abbreviation Sample type Sampling time Altitude Longitude and latitude Urumqi AT-1 M. ovinus June 2021 580–920 m 43°80'N, 87°60'E Kuqa City AT-2 M. ovinus May 2021 930-1,225 m 41°71'N, 82°99'E Yecheng County AT-3 M. ovinus March 2019 1,765 m 37°88'N, 77°41'E Qira County AT-4 M. ovinus May 2023 2,200-3,200 m 36°26'N, 81°03'E Qira County AT-5 Pupae May 2023 2,200-3,200 m 36°26'N, 81°03'E Four M. ovinus (two males and females each) were selected from each sampling site and mixed into one sample. Likewise, four intact-looking pupae were selected and mixed into one sample. The morphological features of M. ovinus and pupae are identified using a Leica stereomicroscope M165 C (Solms, Germany) and photomicrographs were taken. All samples were cleaned in ethanol gradient (70%, 50%, 30%, and 10%) for 30 min at 37℃ and 180 rpm in a culture oscillator at a constant temperature to remove any remaining debris. The samples were then cleaned three more times in sterile distilled water, and finally dried using a filter paper. The DNA was extracted from the processed samples employing the HiPure Soil DNA Kit B (Guangzhou Magen Biotechnology Co., Ltd., China), following the instructions provided. The extracted DNA was stored at -20℃. To prevent any environmental contamination of the samples, the DNA extraction procedures were performed within a biosafety cabinet. Library Preparation and Sequencing Next-generation sequencing libraries were prepared following the manufacturer’s instructions. Genomic DNA (200 µg) was randomly fragmented to an average size of 300–350 bp by Covaris. The fragments were treated using the end-prep enzyme mix for end repair, 5′ phosphorylation, and 3′ adenylated, to add adaptors to both ends. The adaptor-ligated DNA was size-selected by DNA Cleanup beads. Each sample was then amplified by polymerase chain reaction (PCR) for eight cycles using P5 and P7 primers; both primers carry sequences which can anneal with flowcell to perform bridge PCR, while the P7 primer carried a six-base index for multiplexing. The PCR products were cleaned and validated using an Agilent 2100 Bioanalyzer. The qualified libraries were pair-end PE150 sequenced on the Illumina Novaseq 6000 System. Data Analysis The raw image data of sequencing results were identified (base calling) using the software bcl2fastq (v2.17.1.14), and the quality was preliminarily analyzed to obtain the raw data of sequencing samples (Pass Filter Data), and the data were stored in FASTQ (fq) file format. The quality statistics software cutadapt (v1.9.1) was employed to remove junctions and low-quality sequences from the raw data (Pass Filter Data) (primers and junction sequences were removed; bases with quality values less than 20 at both ends were removed; sequences with N base content greater than 10% were removed; minimum reads length of 75 bp were retained). The samples had contamination from the host, the BWA software (v0.7.12) was used to compare with the host genome, and the reads that might be of host origin were filtered out. Assembly analysis was performed based on the optimized Clean Data, using MEGAHIT (v1.1.3) software, suitable for large or complex macro-genomic data assembly and was based on the construction of clean de Bruijn plots for low-memory assembly. Different de bruijin graphs with different K-mer (59, 79, 99, 119, 141) for each sample, were constructed and pre-assembled. Then, the optimal result for that sample was combined with all assembly results as the final assembly result. The coding gene was predicted using Prodigal (v3.02) software, followed by the integration of gene sequences of all samples and further de-redundant processed by the sequence clustering software MMseq2. The non-redundant gene set unigene sequences were obtained by clustering with 95% identity and 95% coverage by default. The number of reads of unigene in each sample on the alignment was obtained using the alignment software SoapAligner (version 2.21) to compare the clean reads obtained after pre-processing to the constructed non-redundant gene set unigene sequences. Then, based on the number of reads on each unigene pair and the length of the gene, the abundance information of unigene in each sample was estimated. The microbial composition of the samples was explored by comparing the unigene sequences with the NR database using diamond. The results of species annotation for each sequence were obtained from the information on taxonomic annotation corresponding to each sequence in the NR database. Combining the results of species annotation of genes and the gene abundance tables, information on each species abundance at each taxonomic level (phylum, order, family, genus, species) could be counted for each sample; next, and for the abundance of a given species in a sample, the abundance was summed for genes annotated as that species. The analysis of α-diversity was based on the species-level results, and the α-diversity indices such as Shannon and Chao1 were calculated to represent the species abundance and community diversity by using random sampling of sequences of samples for leveling, and the rank abundance curve were prepared to reflect the species richness as well as evenness. Results General statistics Sequencing data statistics After calibration, joints and low-quality sequences of the sequencing results were removed from five samples (AT-1, AT-2, AT-3, AT-4, and AT-5), a total of 86,427,864 optimized data sequences (AT-1, 21,989,176; AT-2, 14,431,232; AT-3, 27,058,326; AT-4, 21,176,652; AT-5, 1,772,478,) were obtained, and the respective length of this optimized data was 12,792,106,261 bp (respectively representing 3,272,729,898 bp, 2,132,276,512 bp, 3,997,349,546 bp, 3,130,724,974 bp, and 259,025,331 bp). The mean length of these optimized sequences was 147.66 bp (respectively representing 148.83 bp, 147.75 bp, 147.73 bp, 147.84 bp, and 146.14 bp). Alpha-diversity analysis The AT-2 samples exhibited the highest Ace and Chao1 values, while the AT-4 samples had the highest Shannon and Simpson values. However, as per the guidelines, the Shannon and Simpson values of the AT-2 sample were the most reasonable. The goods coverage value of all the samples was 100%, indicating that the amount of data was sufficient (Table 2 ). Table 2 Alpha-diversity indices and microbial abundance of the samples Sample Ace Chao1 Shannon Simpson Goods coverage AT-1 169.276 178 1.541 0.541 1 AT-2 279.023 288.235 1.646 0.483 1 AT-3 207.135 210.667 1.054 0.333 1 AT-4 253.618 276.333 2.106 0.691 1 AT-5 139.065 139 1.615 0.49 1 Note: The greater the Ace index value and the chao1 index value, the greater the number of species in the community. The higher the Shannon index value, the higher the community diversity. The greater the Simpson index value, the lower the community diversity. The larger the goods coverage index value, the lower the probability that the sequence in the sample is not detected. The rank abundance curve shows the diversity of each sample. A smoother decline and a longer curve indicate a high diversity of the sample, while a fast and steep decline indicates a high proportion of dominant flora and a low sample diversity. The order of the curve span for the five samples was AT-4 > AT-5 > AT-2 > AT-1 > AT-3, as shown in Fig. 2 . This result indicates that samples from the sites AT-4 and AT-5 had the highest microbial diversity. Genus cluster analysis The Genus cluster analysis is shown as a Venn diagram (Fig. 3 ). The respective number of Genus cluster obtained from five samples (AT-1, AT-2, AT-3, AT-4, and AT-5) were 45, 53, 40, 49, and 75. The five samples had 23 highly similar microbial genera, indicating the presence of more of the same microbial populations in the five samples. The AT-5 sample had 40 unique microbial genera, being the highest number of any sample. Microbial population characteristics Microbial characteristics at the phylum level A total of 32 microbial phyla were detected in all samples. Among these, 26, 30, 23, 24, and 20 microbial phyla were detected, respectively, in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial phyla and the community bar plot are presented in Additional File 1: Table S1 and Additional File 2: Fig. S1 A. The dominant phyla were mainly Proteobacteria (respective relative abundance of 94.12%, 91.4%, 97.3%, 92.8%, and 93.9%) and Euglenozoa (respective relative abundance of 5.1%, 7.3%, 1.4%, 6.5%, and 0.0%). Proteobacteria exhibited a clear advantage across samples. Furthermore, Spirochaetes were present only in AT-1 samples, and Thaumarchaeota, Candidatus Moranbacteria, Verrucomicrobia, and Zoopagomycota were present only in AT-2 samples. Characteristics of the microbial genera In all samples, a total of 372 microbial genera were detected, of which 163, 266, 194, 243, and 125 microbial genera were respectively detected in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial genera and the community bar plot is presented in Additional File 1: Table S2 and Additional File 2: Fig. S1 B. The relative abundance of Bartonella in five samples (AT-1 to AT-5, respectively) was 60.9%, 70.2%, 80.3%, 35.3%, and 22.1%. Further, the relative abundance of AT-1, AT-2, and AT-3, was higher than that of AT-4 and AT-5. The relative abundance of Wolbachia in AT-5 was 67.8%, being much higher than that in the remaining four samples. A similar situation was noted for the AT-4 sample, with the relative abundance of Pseudomonas being 40.8%, also much higher than that in the other four samples. A comparison of relative abundance across samples for Arsenophonus (29.0%, 13.2%, 14.8%, 11.1%, and 2.8%) and Trypanosoma (5.1%, 7.2%, 1.4%, 6.5%, and 0.0%) revealed that AT-5 had the lowest relative abundance. The relative abundance of most of the microbial genera, except those mentioned above, was < 1%. Microbial characteristics at the species level A total of 1037 microbial species were detected in all samples, of which 302, 622, 464, 639, and 237 microbial species were, respectively, detected in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial species in terms of microbial community bar plot is shown in Additional File 1: Table S3 and Additional File 2: Fig. S1 C. All the samples had a high relative abundance of Bartonella melophagi (51.3%, 59.1%, 66.7%, 29.6%, and 18.5%), being significantly higher in AT-1, AT-2 and AT-3 than in AT-4 and AT-5. The AT-5 sample had a 47.3% relative abundance of Wolbachia endosymbiont , which was significantly higher than that in the other samples. All the samples had Arsenophonus nasoniae (20.0%, 9.1%, 10.2%, 7.7%, and 2.0%) but AT-1 had the highest relative abundance. The AT-4 sample had a 31.0% relative abundance of Pseudomonas versuta and the rest of the samples was in the range of 0-0.1%. Besides the four bacteria mentioned above, a variety of other bacteria, archaea, fungi, viruses, and eukaryotes were detected in this study, the details of which are presented in Additional File 1: Table S3. Discussion This study was based on the Illumina Novaseq 6000 System for metagenomic sequencing. The research examined the microbial population diversity and disparity in four M. ovinus samples and one pupae sample obtained from Urumqi (AT-1), Kuqa City (AT-2), Yecheng County (AT-3), and Qira County (AT-4 and AT-5), Xinjiang Uygur Autonomous Region, China. metagenomic sequencing can detect less abundant microorganisms that cannot be easily culturable, playing an important role in our research. Given the limitations of metagenomic sequencing, we will analyze the results from a more balanced perspective. The results of the study showed that a total of 1037 species in 32 phyla and 372 genera were detected. After removing the sequences of non-microbial species, 943 microorganisms were detected. Among these, approximately 95 pathogens had varying pathogenicity, representing about 10% of the total. However, most of these pathogens were present in low abundance. The Alpha-diversity indices and the rank abundance curve showed the richest diversity of microbial populations M. ovinus samples from Kuqa City (AT-2) and Qira County (AT-4) contained. Similar to previous studies and as shown by the Venn diagrams, the AT-5 pupae sample, although not the most diverse, has 40 unique genera, which is [ 27 ]. Most of the bacteria, fungi, archaea, spirochetes, chlamydia, viruses, and protozoa in M. ovinus and pupae samples are reported for the first time in this study (see Additional file 1: Table S3 for details). Some microorganisms detected in this study have also been reported previously, such as Bartonella melophagi , Ba. schoenbuchensis, Arsenophonus endosymbiont , Wolbachia endosymbiont , Escherichia coli , Novosphingobium sp., and so on. Symbiotic microorganisms may grow in the gut, body cavity, or cells of insects, potentially causing either positive effects, negative effects, reciprocal effects, or no apparent effect on the host [ 40 ]. The microbial population composition in insects can vary depending on diet, developmental stage, season, and geography [ 41 ]. The most abundant microbial genera detected in the study were Bartonella , Arsenophonus , Wolbachia , Pseudomonas , and Trypanosoma . Bartonella is a gram-negative bacterium transmitted to humans through blood-sucking arthropod vectors, or contact with contaminated animal feces, or on being scratched by infected animals [ 42 ]. Some studies have reported Bartonella sp. with a high rate of infection in M. ovinus samples [ 6 , 11 ]. The last common ancestor of Bartonella was a likely amino acid and cofactor self-reliant gut symbiont that recycles nitrogenous waste products from its insect host; these symbionts can adapt to blood-sucking insects, but may not necessarily adapt to mammalian hosts, causing only opportunistic infections [ 43 ]. We detected Bartonella melophagi , Ba. schoenbuchensis, Ba. bovis , Ba. henselae , Ba. quintana , and so on in our samples. Of these, Ba. Henselae and Ba. quintana cause two serious diseases, cat scratch disease, and trench fever, respectively [ 44 ]. Wolbachia are considered insect symbionts that on the one hand, help their hosts to resist viruses and insecticides, and also aid in addressing some of the nutritional needs of their hosts [ 45 , 46 ], on the other hand, enhance their transmission, induce feminization, male-specific killing, and parthenogenesis of insect hosts [ 47 ]. The relative abundance of Wolbachia was as high as 67.8% in AT-5 pupae samples, much higher than that in adult M. ovinus . As reported by studied by Duan et al., the abundance of Wolbachia in newly hatched and unfed M. ovinus was about 29%, also higher than in the adult abundance [ 27 ]. These values suggest that Wolbachia be supposed to have a greater effect on pupae or larvae than on adults. The genus Arsenophonus is a group of symbiont widely present in a several kinds of insects [ 48 ]. We detected Arsenophonus nasoniae , Ar. endosymbiont , Candidatus Ar senophonus Lipoptenae , and Arsenophonus sp. ENCA in our study. Ar. nasoniae infects people with symptoms of fever and pain [ 49 ]. Most of these symbionts are vertically transmitted from mother to offspring, while a small percent can also be transmitted horizontally [ 50 ]. They affect insects and can also make people or animals sick. Therefore, these symbionts need to be researched further. The bacterium Pseudomonas is one of the most ubiquitous and diverse genera across the world, especially in a wide range of environments, objects, and organisms [ 51 ]. In this study, we detected a variety of Pseudomonas sp., with its abundance in the AT-4 adult M. ovinus samples being much higher than in other samples. This finding may be related to the suitable altitude and climate of the sample location for its growth. Test results included the opportunistic pathogen Pseudomonas aeruginosa , capable of causing serious infections in human respiratory and urinary tracts [ 52 ]. We also detected a variety of bacterial pathogens, such as Brucella abortus , Chlamydia trachomatis , Enterobacter cloacae , Klebsiella pneumoniae , Mycobacterium tuberculosis , Salmonella enterica , Staphylococcus aureus , Yersinia pestis and so on, posing a serious threat to human or animal health. Protozoa have a narrow host range and high specificity, and are thus also used as effective biocontrol agent of pest insects [ 53 ]. In this study, a total of 45 parasitic protozoa were detected, except for Trypanosoma , with a relatively high abundance across samples, while all other protozoa had a relatively low abundance. Until now, no study has reported on the assessment of the interrelationships between M. ovinus and protozoa. The protozoa detected in this study are pathogenic, such as the highly pathogenic protozoa causing Chagas disease ( Trypanosoma cruzi ) or sleeping sickness ( Trypanosoma brucei ) in humans [ 54 ], T richomonas vaginalis , which causes diseases of the human reproductive and urinary systems [ 55 ], and Plasmodium falciparum , which causes severe anemia and cerebral malaria [ 56 ]. To date, only five studies have reported the detection of viruses from M. ovinus [ 12 , 20 , 21 , 22 , 23 ]. In the current study, only the Orf virus and multiple phages were detected. The Orf virus is a DNA virus of genus Parapoxvirus , a highly contagious zoonotic disease causing a highly contagious vesiculo ulcerative pustular infection [ 57 ]. Phages present in M. ovinus regulate the bacterial community. The absence of phages may cause an imbalance in the bacterial community in M. ovinus and adversely affect its health. In this study only 11 phage species, including Wolbachia phage WO, Escherichia phage L AB-2017, and Staphylococcus phage VB-SauS-SA2, with very low abundance, were detected. Microorganisms that parasitize insect hosts can positively affect the host, such as providing nutrients or enhancing the fitness of the host while obtaining nutrients for themselves. The microorganisms may also be pathogenic to the insect host, reducing the fitness or causing its death. Therefore, studying microbial community diversity in M. ovinus and determining their roles and functions will make it possible to efficiently investigate a way to manage pests. In this study, multiple pathogens were found in only one or two locations, suggesting that some pathogens may be endemic. During blood-sucking, M. ovinus may lead to epidemics of these pathogens in sheep, which in turn may threaten public health safety or the stability of the sheep industry economy. In this study, a variety of microorganisms were detected, and the data on the microbial diversity of M. ovinus in Xinjiang were enriched. However, the data on the microbial diversity of M. ovinus in various places is still insignificant to. In conclusion, our study has implications regarding veterinary and public health safety. Based on the pathogen diversity, we need to assess their potential risk of M. ovinus to animal husbandry and public health and control its infestations. The relationship, between microorganisms and M. ovinus and its transmission and function can be used to develop methods for biological prevention and control of the pests. Conclusions In this study, 32 microbial phyla were detected in sheep ticks in Xinjiang, China, with Proteobacteria being the dominant phylum. Bartonella , Wolbachia , Pseudomonas , and Arsenophonus were the dominant genera among the 372 genera detected. After eliminating non-microbial species, 943 microorganisms, containing nearly 100 pathogens were detected. This is the first study to report most of the bacteria (e.g., Bartonella bovis and Arsenophonus nasoniae ), fungi (e.g., Saitoella complicata ), viruses (e.g., Orf virus and Wolbachia phage WO), and protozoa (e.g., Trypanosoma theileri and Trichomonas vaginalis ) in M. ovinus . Abbreviations USD United States dollar PCR Polymerase Chain Reaction NR database Non-Redundant Protein Database. Declarations Acknowledgements We thank members of our laboratories for fruitful discussions. Funding This study was supported by grants awarded by the National Natural Science Foundation of China (Granted No. 32160841 and 31960705), Key Laboratory of Tarim Animal Husbandry Science and Technology, Xinjiang Production & Construction Group (HS201802), and Tarim University Graduate Student Research Innovation Project (TDGRI202240). Availability of data and materials The raw tags have been deposited in the Sequence Read Archive (SRA) from the NCBI under BioProject accession PRJNA1074431. The individual run files received accession numbers SRR27908141, SRR27908142, SRR27908143, SRR27908144 and SRR27908145. (A link to the BioProject and associated SRA metadata is ready to copy and share with reviewers via your publisher. https://dataview.ncbi.nlm.nih.gov/object/PRJNA1074431?reviewer=sljjb8knean2cg8jidfsk7n3u1) Authors’ contributions JW and KH provided the research idea. XQ and KZ performed the collection and assembly of data. XZ and NX performed the experiments. QF and LS performed the data analysis and interpretation. KH wrote the manuscript. JW handled the critical revision of the article. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version. Ethics approval and consent to participate No ethics approval was required as ectoparasite collection from the sheep was part of the routine medical procedure for rescued animals. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests References Small RW. A review of Melophagus ovinus (L.), the sheep ked. Vet Parasitol. 2005; 130:141–55. Zhao L, Wang J, Ding Y, Li K, He B, Li F, et al. Theileria ovis (Piroplasmida: Theileriidae) Detected in Melophagus ovinus (Diptera: Hippoboscoidea) and Ornithodoros lahorensis (Ixodida: Argasidae) Removed From Sheep in Xinjiang, China. J Med Entomol. 2020; 57:631–635. Seyoum Z, Tadesse T, Addisu A. Ectoparasites Prevalence in Small Ruminants in and around Sekela, Amhara Regional State, Northwest Ethiopia. J Vet Med. 2015;2015:216085. Flores-Mendoza C, Loyola S, Jiang J, Farris CM, Mullins K, Estep AS, et al. 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Major Causes of Calf and Lamb Mortality and Morbidity and Associated Risk Factors in the Mixed Crop-Livestock Production System in Jamma District, South Wollo, Ethiopia. Vet Med Int. 2021;2021:6689154. Sertse T, Wossene A. Effect of ectoparasites on quality of pickled skins and their impact on the tanning industries in Amhara regional state, Ethiopia. Small Ruminant Res. 2007;69:55–61. Luedke AJ, Jochim MM, Bowne JG. Preliminary bluetongue Transmission with the sheep ked Melophagus ovinus (L.). Can J Comp Med Vet Sci. 1965;29:229–31. Setién Á A, Baltazar AG, Leyva IO, Rojas MS, Koldenkova VP, García MP, et al. Ectoparasitic hematophagous dipters: potential reservoirs of dengue virus? Gac Med Mex. 2017;153(Supl. 2):S82-S90. Liu YH, He B, Li KR, Li F, Zhang LY, Li XQ, et al. First report of border disease virus in Melophagus ovinus (sheep ked) collected in Xinjiang, China. Plos One. 2019;14:e0221435. Liu YH, Ma YM, Tian HO, Yang B, Han WX, Zhao WH, et al. 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Insect-Symbiont Gene Expression in the Midgut Bacteriocytes of a Blood-Sucking Parasite. Genome Biol Evol. 2020;12:429–442. Chu CY, Jiang BG, Qiu EC, Zhang F, Zuo SQ, Yang H, et al. Borrelia burgdorferi sensu lato in sheep keds ( Melophagus ovinus ), Tibet, China. Vet Microbiol. 2011;149:526–9. Pavilanis V, Duval L, Foley AR, L'Heureux M. An epidemic of Q fever at Princeville, Quebec. Can J Public Health. 1958;49:520–9. Liu D, Wang YZ, Zhang H, Liu ZQ, Wureli HZ, Wang SW, et al. First report of Rickettsia raoultii and R. slovaca in Melophagus ovinus , the sheep ked. Parasit Vectors. 2016;9:600. Hao L, Yuan D, Li S, Jia T, Guo L, Hou W, et al. Detection of Theileria spp. in ticks, sheep keds ( Melophagus ovinus ), and livestock in the eastern Tibetan Plateau, China. Parasitol Res. 2020;119:2641–2648. Yadav KK, Datta S, Naglot A, Bora A, Hmuaka V, Bhagyawant S, et al. Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India. Plos One. 2016;11:e0167409. Wang Y, Gilbreath TM III, Kukutla P, Yan G, Xu J. Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya. Plos One. 2011;6:e24767. Telleria EL, Martins-da-Silva A, Tempone AJ, Traub-Csekö YM. Leishmania, microbiota and sand fly immunity. Parasitology. 2018;145:1336–1353. Douglas AE. Multiorganismal insects: diversity and function of resident microorganisms. Annu Rev Entomol. 2015;60:17–34. Jing TZ, Qi FH, Wang ZY. Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision? Microbiome. 2020;8:38. Bezerra-Santos MA, Otranto D. Keds, the enigmatic flies and their role as vectors of pathogens. Acta Trop. 2020;209:105521. Zhang Q, Zhou Q, Han S, Li Y, Wang Y, He H. The genome of sheep ked ( Melophagus ovinus ) reveals potential mechanisms underlying reproduction and narrower ecological niches. BMC Genomics. 2023;24:54 Hosokawa T, Fukatsu T. Relevance of microbial symbiosis to insect behavior. Curr Opin Insect Sci. 2020;39:91–100. Bascuñán P, Niño-Garcia JP, Galeano-Castañeda Y, Serre D, Correa MM. Factors shaping the gut bacterial community assembly in two main Colombian malaria vectors. Microbiome. 2018;6:148. Zhang L, Peng Q, Gu XL, Su WQ, Cao XQ, Zhou CM, et al. Host specificity and genetic diversity of Bartonella in rodents and shrews from Eastern China. Transbound Emerg Dis. 2022;69:3906–3916. Segers FH, Kešnerová L, Kosoy M, Engel P. Genomic changes associated with the evolutionary transition of an insect gut symbiont into a blood-borne pathogen. The ISME journal. 2017;11:1232–1244. Minnick MF, Anderson BE. Chapter 105 - Bartonella . In: Tang Y-W, Sussman M, Liu D, Poxton I, Schwartzman J, editors. Molecular Medical Microbiology (Second Edition). Boston: Academic Press; 2015. p. 1911-39. Hedges LM, Brownlie JC, O'Neill SL, Johnson KN. Wolbachia and virus protection in insects. Science. 2008;322:702. Miller WJ. Bugs in transition: the dynamic world of Wolbachia in insects. PLoS Genet. 2013;9:e1004069. Werren JH, Baldo L, Clark ME. Wolbachia : master manipulators of invertebrate biology. Nat Rev Microbiol. 2008;6:741–51. Nováková E, Hypsa V, Moran NA. Arsenophonus , an emerging clade of intracellular symbionts with a broad host distribution. BMC Microbiol. 2009;9:143. Liew KC, Graves S, Croft L, Brettell LE, Cook J, Botes J, et al. First human case of infection with Arsenophonus nasoniae , the male killer insect pathogen. Pathology. 2022;54:664–666. Parratt SR, Frost CL, Schenkel MA, Rice A, Hurst GD, King KC. Superparasitism Drives Heritable Symbiont Epidemiology and Host Sex Ratio in a Wasp. PLoS Pathog. 2016;12:e1005629. Peix A, Ramírez-Bahena MH, Velázquez E. The current status on the taxonomy of Pseudomonas revisited: An update. Infect Genet Evol. 2018;57:106–16. Silby MW, Winstanley C, Godfrey SA, Levy SB, Jackson RW. Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev. 2011;35:652–80. Gurung K, Wertheim B, Falcao Salles J. The microbiome of pest insects: it is not just bacteria. Entomol Exp Appl. 2019;167:156–170. Calzolari M, Rugna G, Clementi E, Carra E, Pinna M, Bergamini F, et al. Isolation of a Trypanosome Related to Trypanosoma theileri (Kinetoplastea: Trypanosomatidae) from Phlebotomus perfiliewi (Diptera: Psychodidae). Biomed Res Int. 2018;2018:2597074. Kissinger P. Trichomonas vaginalis : a review of epidemiologic, clinical and treatment issues. BMC Infect Dis. 2015;15:307. Maier AG, Matuschewski K, Zhang M, Rug M. Plasmodium falciparum . Trends Parasitol. 2019;35:481–482. Bergqvist C, Kurban M, Abbas O. Orf virus infection. Rev Med Virol. 2017;27. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1TableS1S3.docx Additional file 1: Table S1. The absolute abundance of 32 microbial phyla detected in five samples from different parts of China. Table S2. The absolute abundance of 372 microbial genera in the five samples. Table S3. The absolute abundance of 1037 microbial species in the above-mentioned five samples. Additionalfile2Fig.S1.jpg Additional file 2: Fig. S1. A bar plot presenting the microbial community detected in the five samples (A Microbial phylum; B Microbial Genus; C Microbial species). Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Frontiers in Veterinary Science → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3990667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275298916,"identity":"40d304b5-624d-49bd-8fcf-7a5fa36df2ba","order_by":0,"name":"Kaijun Huang","email":"","orcid":"","institution":"College of Animal Science and Technology, Tarim University, Alar, Xinjiang, 843300","correspondingAuthor":false,"prefix":"","firstName":"Kaijun","middleName":"","lastName":"Huang","suffix":""},{"id":275298917,"identity":"8b08837a-6d41-49bd-9cc4-7d874382ae41","order_by":1,"name":"Xing Zhang","email":"","orcid":"","institution":"College of 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Tarim University, Alar, Xinjiang, 843300","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Xiong","suffix":""},{"id":275298921,"identity":"f950e4c0-52bf-4e30-9457-486bca0fa064","order_by":5,"name":"Xiaoqing Zhao","email":"","orcid":"","institution":"College of Animal Science and Technology, Tarim University, Alar, Xinjiang, 843300","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Zhao","suffix":""},{"id":275298922,"identity":"89f392d3-c59d-4294-9f5f-496c5f91ddf5","order_by":6,"name":"Kun Zhou","email":"","orcid":"","institution":"College of Animal Science and Technology, Tarim University, Alar, Xinjiang, 843300","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Zhou","suffix":""},{"id":275298923,"identity":"dc86008f-c9f1-4f44-b084-2cb09a023720","order_by":7,"name":"Junyuan 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10:32:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3990667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3990667/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3389/fvets.2024.1462772","type":"published","date":"2024-12-04T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51809819,"identity":"5081425c-0e13-4d38-b213-c140519b53eb","added_by":"auto","created_at":"2024-02-29 12:11:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":702969,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of adult and pupae of \u003cem\u003eMelophagus ovinus\u003c/em\u003e(scale bar: 1 mm). \u003cstrong\u003eA\u003c/strong\u003e Back view and \u003cstrong\u003eB\u003c/strong\u003e Ventral view of the female. \u003cstrong\u003eC\u003c/strong\u003e Back view and \u003cstrong\u003eD\u003c/strong\u003e Ventral view of the male. \u003cstrong\u003eE\u003c/strong\u003ePupae of \u003cem\u003eM. ovinus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.1PhotomicrographsofadultandpupaeofMelophagusovinusscalebar1mm.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/b0ae78e2349755d11052f330.jpg"},{"id":51809824,"identity":"3862f95a-755d-46c3-b859-2ad48e5272d7","added_by":"auto","created_at":"2024-02-29 12:11:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":286747,"visible":true,"origin":"","legend":"\u003cp\u003eThe rank abundance curve. The X-axis shows the microbial species in the order of highest to lowest relative abundance, and the Y-axis presents the relative abundance of the microbial species. Curves of different colors represent different samples\u003c/p\u003e","description":"","filename":"Fig.2Therankabundancecurve.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/ad161623c195e82982cb3786.jpg"},{"id":51809821,"identity":"239cf6bf-d645-4bd2-8e94-8a7705c26c3f","added_by":"auto","created_at":"2024-02-29 12:11:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":702736,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagrams of microbial abundance in five samples based on Genus\u003c/p\u003e","description":"","filename":"Fig.3VenndiagramsofmicrobialabundanceinfivesamplesbasedonGenus.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/3accbbf06480e76fe5ffca17.jpg"},{"id":70785493,"identity":"d9879579-f32f-47a3-93b1-984e321d970c","added_by":"auto","created_at":"2024-12-06 16:41:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2382158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/81b4d49e-4aad-47bc-9458-46b11572f072.pdf"},{"id":51809927,"identity":"24de1c95-1f93-4e88-986e-f90824235509","added_by":"auto","created_at":"2024-02-29 12:19:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":190668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1. \u003c/strong\u003eThe absolute abundance of 32 microbial phyla detected in five samples from different parts of China. \u003cstrong\u003eTable S2. \u003c/strong\u003eThe absolute abundance of 372 microbial genera in the five samples.\u003cstrong\u003e Table S3. \u003c/strong\u003eThe absolute abundance of 1037 microbial species in the above-mentioned five samples.\u003c/p\u003e","description":"","filename":"Additionalfile1TableS1S3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/5364357144584ba38fdde523.docx"},{"id":51809822,"identity":"afc17065-d93b-4d68-8a90-85469e7b4b2b","added_by":"auto","created_at":"2024-02-29 12:11:40","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1176484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Fig. S1. \u003c/strong\u003eA bar plot\u003cstrong\u003e \u003c/strong\u003epresenting the\u003cstrong\u003e \u003c/strong\u003emicrobial community detected in the five samples (A Microbial phylum; B Microbial Genus; C Microbial species).\u003c/p\u003e","description":"","filename":"Additionalfile2Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3990667/v1/d7fcb31448ce8f2ba53a5274.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"First metagenomic sequencing for the analysis of microbial community population of Melophagus ovinus and pupae in Xinjiang, China","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eMelophagus ovinus\u003c/em\u003e is a blood-sucking parasite that parasitizes primarily on the body surface of sheep. This ectoparasite lacks wings and has a surface covered with bristles, robust mouthparts, and three pairs of sharp grappling hooks. \u003cem\u003eM. ovinus\u003c/em\u003e is a member of the Hippoboscidae (Diptera, Hippoboscoidea) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; it also parasitizes the body surfaces of mammals, including Tibetan antelope [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], goats [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], rabbits [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], donkeys [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and so on. The life cycle of this parasite consists of three distinct stages: larva, pupa, and wingless adult. Shortly after birth, female \u003cem\u003eM. ovinus\u003c/em\u003e mates and produces offspring, giving birth to 12\u0026ndash;15 offspring in her lifetime [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The geographic distribution of \u003cem\u003eM. ovinus\u003c/em\u003e is very wide and it has been reported in Europe (Slovakia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], France [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], England [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Hungary [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Croatia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Czechia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], Poland [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Russia [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]), Asia (Turkey [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and China [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]), South America (Peru [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]), North America (USA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]), Oceania (Australia [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and New Zealand [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]) and Africa (Algeria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Ethiopia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eThe parasitization of sheep with large amounts of \u003cem\u003eM. ovinus\u003c/em\u003e results in two serious consequences. First, by biting and sucking blood, it makes the skin of sheep itchy and painful, leading to skin damage, wool loss, agitation, anemia, and weight loss. More severe cases can be secondary to pathogenic microbial infections and cutaneous myiasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The infestation can lead to a loss in the quality and production from sheep products, such as skin, wool, meat, and milk, adversely affecting the economy of the fur and livestock industries. Massive infections of \u003cem\u003eM. ovinus\u003c/em\u003e led to a loss of 1.6\u0026nbsp;million USD by two Ethiopian tanneries over two years [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMore importantly, \u003cem\u003eM. ovinus\u003c/em\u003e carries a variety of pathogenic microorganisms that infect the host in the process of sucking blood, such as viruses (e.g., Bluetongue virus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Border disease virus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and African swine fever virus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]), \u003cem\u003eAcinetobacter\u003c/em\u003e spp. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (e.g., \u003cem\u003eAc. lwoffii\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]), \u003cem\u003eAnaplasma\u003c/em\u003e spp. (e.g., \u003cem\u003eAn. ovis\u003c/em\u003e, \u003cem\u003eAn. Phagocytophilum\u003c/em\u003e, and \u003cem\u003eAn. bovis\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]), \u003cem\u003eArsenophonus\u003c/em\u003e spp. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] (e.g., \u003cem\u003eAr. melophagi\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]), \u003cem\u003eBartonella\u003c/em\u003e spp. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] (e.g., \u003cem\u003eBa. schoenbuchensis\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], \u003cem\u003eBa. chomelii\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and \u003cem\u003eBa. melophagi\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]), \u003cem\u003eBorrelia\u003c/em\u003e spp. (e.g., \u003cem\u003eBo. garinii\u003c/em\u003e, \u003cem\u003eBo. spirochetes\u003c/em\u003e belonging to the \u003cem\u003eBo. valaisiana\u003c/em\u003e-related group [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and \u003cem\u003eBo. burgdorferi\u003c/em\u003e sensu lato [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]), \u003cem\u003eCoxiella\u003c/em\u003e spp. (e.g., \u003cem\u003eCo. burnetii\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]), \u003cem\u003eRickettsia\u003c/em\u003e spp. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (e.g., \u003cem\u003eRi. raoultii, Ri. slovaca\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eRi. melophagi\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003eRi. massiliae\u003c/em\u003e, and \u003cem\u003eCandidatus\u003c/em\u003e Rickettsia barbariae [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]), \u003cem\u003eTheileria\u003c/em\u003e spp. (e.g., \u003cem\u003eTh. ovis\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and \u003cem\u003eTh. Luwenshuni\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]), \u003cem\u003eTrypanosoma\u003c/em\u003e spp. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] (e.g., \u003cem\u003eTr. theodori\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and \u003cem\u003eTr. melophagium\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]), \u003cem\u003eWolbachia\u003c/em\u003e sp. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], etc. Most studies on \u003cem\u003eM. ovinus\u003c/em\u003e have focused specifically on one to three pathogens. Concurrently, few studies have also detected and identified the types of viruses they carry or analyzed their microbial communities.\u003c/p\u003e \u003cp\u003eMicroorganisms colonize the body surface, intestine, hemocoel, and even cells of all insects, that serve as vectors for microorganisms. Their microbial composition and abundance can be altered by a variety of conditions, including insect species, sex, developmental stage, blood-sucking behavior, survival strategies, and geographic location [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Among these conditions, feeding on the blood reduces the bacterial diversity of the insect gut [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Insects and the microorganisms they carry may be in mutually beneficial symbiotic, parasitic, and competitive relationships. For example, microorganisms promote insect health, synthesize toxins or modify the insect immune system to protect it from pathogens, parasitize insects, and attack other hosts at opportune times [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The most important function of microorganisms in symbiotic association with insects is the provision of nutrients, digestion and detoxification [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe study of the microbial community composition and abundance of \u003cem\u003eM. ovinus\u003c/em\u003e contributes to scientific knowledge concerning its biology and epidemiology. Metagenomic sequencing involves the high-throughput sequencing of the genomes of microbial communities in a sample, and facilitates the study of microbial population structure. This technique is free from the limitations of microbial isolation and pure culture, and can detect microorganisms in trace numbers, providing an effective tool for studying microbial communities in samples. Currently, only four studies have reported using high-throughput sequencing to analyze the microbial composition and diversity in \u003cem\u003eM. ovinus\u003c/em\u003e. In 2017, Duan et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] researched the characterization of midgut microbial populations in male and female \u003cem\u003eM. ovinus\u003c/em\u003e, and in 2020, their team [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] extracted DNA from fully satiated, newly hatched and unfed female \u003cem\u003eM. ovinus\u003c/em\u003e the midgut and whole body for bacterial community studies. Later in 2021, Litov et al. studied the virome of \u003cem\u003eM. ovinus\u003c/em\u003e and annotated the full genomes of five novel viruses, and also proved that the Aksy-Durug Melophagus sigma virus can replicate in mammalian cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In 2022, Liu et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] studied adult \u003cem\u003eM. ovinus\u003c/em\u003e from three regions of Tibet and discovered for the first time, 23 bacterial genera and multiple DNA viruses. In 2023, Zhang et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] sequenced, assembled, and annotated the full genome of \u003cem\u003eM. ovinus\u003c/em\u003e. They also discovered that contractions and losses of sensory receptors and vision-associated Rhodopsin genes were significant in \u003cem\u003eM. ovinus\u003c/em\u003e, and could contribute to the removal of host-sequence contamination during the high-throughput sequencing data processing.\u003c/p\u003e \u003cp\u003eIn this study, we employed the Illumina Novaseq 6000 System to metagenomic sequencing and analysis of the microbial composition and abundance in adult and pupae of \u003cem\u003eM. ovinus\u003c/em\u003e from four regions in Xinjiang, China. This is the first report of the use of a technique has been used to study \u003cem\u003eM. ovinus\u003c/em\u003e in the area surrounding the Taklamakan Desert, the second most mobile desert in the world, as well as the first study of microbial diversity in pupae. The blood-feeding behavior of insects significantly affects microbial composition and abundance within and outside their body. Thus, it is of great interest to study and analyze the microbial diversity of adult \u003cem\u003eM. ovinus\u003c/em\u003e and pupae simultaneously from different regions. This study aims to provide scientific data for the discovery of new pathogens, assessment of their harm to public health and industrial economy, formulation of measures to prevent and control arboviruses spread, and the development of biological control methods.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and DNA extraction\u003c/h2\u003e \u003cp\u003e \u003cem\u003eM. ovinus\u003c/em\u003e adults and pupae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were collected from four locations in Xinjiang, China, in 2019 to 2023 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All hosts were sheep. \u003cem\u003eM. ovinus\u003c/em\u003e was collected during the treatment of sheep for ectoparasites. The samples were stored in a -80℃ refrigerator until use. \u003cem\u003eM. ovinus\u003c/em\u003e and its pupae were identified using morphological and molecular biology methods. For better description, the samples will be hence referred to by the abbreviations AT-1, AT-2, AT-3, AT-4, and AT-5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetailed sample information for \u003cem\u003eMelophagus ovinus\u003c/em\u003e and pupae\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample abbreviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSample type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSampling time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAltitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLongitude and latitude\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrumqi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM. ovinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJune 2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e580\u0026ndash;920 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u0026deg;80'N, 87\u0026deg;60'E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKuqa City\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM. ovinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e930-1,225 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41\u0026deg;71'N, 82\u0026deg;99'E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYecheng County\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM. ovinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,765 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37\u0026deg;88'N, 77\u0026deg;41'E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQira County\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM. ovinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,200-3,200 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36\u0026deg;26'N, 81\u0026deg;03'E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQira County\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePupae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,200-3,200 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36\u0026deg;26'N, 81\u0026deg;03'E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFour \u003cem\u003eM. ovinus\u003c/em\u003e (two males and females each) were selected from each sampling site and mixed into one sample. Likewise, four intact-looking pupae were selected and mixed into one sample. The morphological features of \u003cem\u003eM. ovinus\u003c/em\u003e and pupae are identified using a Leica stereomicroscope M165 C (Solms, Germany) and photomicrographs were taken. All samples were cleaned in ethanol gradient (70%, 50%, 30%, and 10%) for 30 min at 37℃ and 180 rpm in a culture oscillator at a constant temperature to remove any remaining debris. The samples were then cleaned three more times in sterile distilled water, and finally dried using a filter paper.\u003c/p\u003e \u003cp\u003eThe DNA was extracted from the processed samples employing the HiPure Soil DNA Kit B (Guangzhou Magen Biotechnology Co., Ltd., China), following the instructions provided. The extracted DNA was stored at -20℃. To prevent any environmental contamination of the samples, the DNA extraction procedures were performed within a biosafety cabinet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLibrary Preparation and Sequencing\u003c/h2\u003e \u003cp\u003eNext-generation sequencing libraries were prepared following the manufacturer\u0026rsquo;s instructions. Genomic DNA (200 \u0026micro;g) was randomly fragmented to an average size of 300\u0026ndash;350 bp by Covaris. The fragments were treated using the end-prep enzyme mix for end repair, 5\u0026prime; phosphorylation, and 3\u0026prime; adenylated, to add adaptors to both ends. The adaptor-ligated DNA was size-selected by DNA Cleanup beads. Each sample was then amplified by polymerase chain reaction (PCR) for eight cycles using P5 and P7 primers; both primers carry sequences which can anneal with flowcell to perform bridge PCR, while the P7 primer carried a six-base index for multiplexing. The PCR products were cleaned and validated using an Agilent 2100 Bioanalyzer. The qualified libraries were pair-end PE150 sequenced on the Illumina Novaseq 6000 System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThe raw image data of sequencing results were identified (base calling) using the software bcl2fastq (v2.17.1.14), and the quality was preliminarily analyzed to obtain the raw data of sequencing samples (Pass Filter Data), and the data were stored in FASTQ (fq) file format. The quality statistics software cutadapt (v1.9.1) was employed to remove junctions and low-quality sequences from the raw data (Pass Filter Data) (primers and junction sequences were removed; bases with quality values less than 20 at both ends were removed; sequences with N base content greater than 10% were removed; minimum reads length of 75 bp were retained). The samples had contamination from the host, the BWA software (v0.7.12) was used to compare with the host genome, and the reads that might be of host origin were filtered out.\u003c/p\u003e \u003cp\u003eAssembly analysis was performed based on the optimized Clean Data, using MEGAHIT (v1.1.3) software, suitable for large or complex macro-genomic data assembly and was based on the construction of clean de Bruijn plots for low-memory assembly. Different de bruijin graphs with different K-mer (59, 79, 99, 119, 141) for each sample, were constructed and pre-assembled. Then, the optimal result for that sample was combined with all assembly results as the final assembly result.\u003c/p\u003e \u003cp\u003e The coding gene was predicted using Prodigal (v3.02) software, followed by the integration of gene sequences of all samples and further de-redundant processed by the sequence clustering software MMseq2. The non-redundant gene set unigene sequences were obtained by clustering with 95% identity and 95% coverage by default. The number of reads of unigene in each sample on the alignment was obtained using the alignment software SoapAligner (version 2.21) to compare the clean reads obtained after pre-processing to the constructed non-redundant gene set unigene sequences. Then, based on the number of reads on each unigene pair and the length of the gene, the abundance information of unigene in each sample was estimated.\u003c/p\u003e \u003cp\u003eThe microbial composition of the samples was explored by comparing the unigene sequences with the NR database using diamond. The results of species annotation for each sequence were obtained from the information on taxonomic annotation corresponding to each sequence in the NR database. Combining the results of species annotation of genes and the gene abundance tables, information on each species abundance at each taxonomic level (phylum, order, family, genus, species) could be counted for each sample; next, and for the abundance of a given species in a sample, the abundance was summed for genes annotated as that species.\u003c/p\u003e \u003cp\u003eThe analysis of α-diversity was based on the species-level results, and the α-diversity indices such as Shannon and Chao1 were calculated to represent the species abundance and community diversity by using random sampling of sequences of samples for leveling, and the rank abundance curve were prepared to reflect the species richness as well as evenness.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneral statistics\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eSequencing data statistics\u003c/h2\u003e \u003cp\u003eAfter calibration, joints and low-quality sequences of the sequencing results were removed from five samples (AT-1, AT-2, AT-3, AT-4, and AT-5), a total of 86,427,864 optimized data sequences (AT-1, 21,989,176; AT-2, 14,431,232; AT-3, 27,058,326; AT-4, 21,176,652; AT-5, 1,772,478,) were obtained, and the respective length of this optimized data was 12,792,106,261 bp (respectively representing 3,272,729,898 bp, 2,132,276,512 bp, 3,997,349,546 bp, 3,130,724,974 bp, and 259,025,331 bp). The mean length of these optimized sequences was 147.66 bp (respectively representing 148.83 bp, 147.75 bp, 147.73 bp, 147.84 bp, and 146.14 bp).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAlpha-diversity analysis\u003c/h2\u003e \u003cp\u003eThe AT-2 samples exhibited the highest Ace and Chao1 values, while the AT-4 samples had the highest Shannon and Simpson values. However, as per the guidelines, the Shannon and Simpson values of the AT-2 sample were the most reasonable. The goods coverage value of all the samples was 100%, indicating that the amount of data was sufficient (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAlpha-diversity indices and microbial abundance of the samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAce\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChao1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGoods coverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e169.276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e279.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e288.235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e207.135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e210.667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e253.618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e276.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e139.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: The greater the Ace index value and the chao1 index value, the greater the number of species in the community. The higher the Shannon index value, the higher the community diversity. The greater the Simpson index value, the lower the community diversity. The larger the goods coverage index value, the lower the probability that the sequence in the sample is not detected.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe rank abundance curve shows the diversity of each sample. A smoother decline and a longer curve indicate a high diversity of the sample, while a fast and steep decline indicates a high proportion of dominant flora and a low sample diversity. The order of the curve span for the five samples was AT-4\u0026thinsp;\u0026gt;\u0026thinsp;AT-5\u0026thinsp;\u0026gt;\u0026thinsp;AT-2\u0026thinsp;\u0026gt;\u0026thinsp;AT-1\u0026thinsp;\u0026gt;\u0026thinsp;AT-3, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This result indicates that samples from the sites AT-4 and AT-5 had the highest microbial diversity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGenus cluster analysis\u003c/h2\u003e \u003cp\u003eThe Genus cluster analysis is shown as a Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The respective number of Genus cluster obtained from five samples (AT-1, AT-2, AT-3, AT-4, and AT-5) were 45, 53, 40, 49, and 75. The five samples had 23 highly similar microbial genera, indicating the presence of more of the same microbial populations in the five samples. The AT-5 sample had 40 unique microbial genera, being the highest number of any sample.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial population characteristics\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eMicrobial characteristics at the phylum level\u003c/h2\u003e \u003cp\u003eA total of 32 microbial phyla were detected in all samples. Among these, 26, 30, 23, 24, and 20 microbial phyla were detected, respectively, in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial phyla and the community bar plot are presented in Additional File 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Additional File 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A. The dominant phyla were mainly Proteobacteria (respective relative abundance of 94.12%, 91.4%, 97.3%, 92.8%, and 93.9%) and Euglenozoa (respective relative abundance of 5.1%, 7.3%, 1.4%, 6.5%, and 0.0%). Proteobacteria exhibited a clear advantage across samples. Furthermore, Spirochaetes were present only in AT-1 samples, and Thaumarchaeota, Candidatus Moranbacteria, Verrucomicrobia, and Zoopagomycota were present only in AT-2 samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of the microbial genera\u003c/h2\u003e \u003cp\u003eIn all samples, a total of 372 microbial genera were detected, of which 163, 266, 194, 243, and 125 microbial genera were respectively detected in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial genera and the community bar plot is presented in Additional File 1: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Additional File 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e B. The relative abundance of \u003cem\u003eBartonella\u003c/em\u003e in five samples (AT-1 to AT-5, respectively) was 60.9%, 70.2%, 80.3%, 35.3%, and 22.1%. Further, the relative abundance of AT-1, AT-2, and AT-3, was higher than that of AT-4 and AT-5. The relative abundance of \u003cem\u003eWolbachia\u003c/em\u003e in AT-5 was 67.8%, being much higher than that in the remaining four samples. A similar situation was noted for the AT-4 sample, with the relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e being 40.8%, also much higher than that in the other four samples. A comparison of relative abundance across samples for \u003cem\u003eArsenophonus\u003c/em\u003e (29.0%, 13.2%, 14.8%, 11.1%, and 2.8%) and \u003cem\u003eTrypanosoma\u003c/em\u003e (5.1%, 7.2%, 1.4%, 6.5%, and 0.0%) revealed that AT-5 had the lowest relative abundance. The relative abundance of most of the microbial genera, except those mentioned above, was \u0026lt;\u0026thinsp;1%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial characteristics at the species level\u003c/h2\u003e \u003cp\u003eA total of 1037 microbial species were detected in all samples, of which 302, 622, 464, 639, and 237 microbial species were, respectively, detected in AT-1, AT-2, AT-3, AT-4, and AT-5. The absolute abundance of these microbial species in terms of microbial community bar plot is shown in Additional File 1: Table S3 and Additional File 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e C. All the samples had a high relative abundance of \u003cem\u003eBartonella melophagi\u003c/em\u003e (51.3%, 59.1%, 66.7%, 29.6%, and 18.5%), being significantly higher in AT-1, AT-2 and AT-3 than in AT-4 and AT-5. The AT-5 sample had a 47.3% relative abundance of \u003cem\u003eWolbachia endosymbiont\u003c/em\u003e, which was significantly higher than that in the other samples. All the samples had \u003cem\u003eArsenophonus nasoniae\u003c/em\u003e (20.0%, 9.1%, 10.2%, 7.7%, and 2.0%) but AT-1 had the highest relative abundance. The AT-4 sample had a 31.0% relative abundance of \u003cem\u003ePseudomonas versuta\u003c/em\u003e and the rest of the samples was in the range of 0-0.1%. Besides the four bacteria mentioned above, a variety of other bacteria, archaea, fungi, viruses, and eukaryotes were detected in this study, the details of which are presented in Additional File 1: Table S3.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study was based on the Illumina Novaseq 6000 System for metagenomic sequencing. The research examined the microbial population diversity and disparity in four \u003cem\u003eM. ovinus\u003c/em\u003e samples and one pupae sample obtained from Urumqi (AT-1), Kuqa City (AT-2), Yecheng County (AT-3), and Qira County (AT-4 and AT-5), Xinjiang Uygur Autonomous Region, China. metagenomic sequencing can detect less abundant microorganisms that cannot be easily culturable, playing an important role in our research. Given the limitations of metagenomic sequencing, we will analyze the results from a more balanced perspective. The results of the study showed that a total of 1037 species in 32 phyla and 372 genera were detected. After removing the sequences of non-microbial species, 943 microorganisms were detected. Among these, approximately 95 pathogens had varying pathogenicity, representing about 10% of the total. However, most of these pathogens were present in low abundance.\u003c/p\u003e \u003cp\u003eThe Alpha-diversity indices and the rank abundance curve showed the richest diversity of microbial populations \u003cem\u003eM. ovinus\u003c/em\u003e samples from Kuqa City (AT-2) and Qira County (AT-4) contained. Similar to previous studies and as shown by the Venn diagrams, the AT-5 pupae sample, although not the most diverse, has 40 unique genera, which is [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Most of the bacteria, fungi, archaea, spirochetes, chlamydia, viruses, and protozoa in \u003cem\u003eM. ovinus\u003c/em\u003e and pupae samples are reported for the first time in this study (see Additional file 1: Table S3 for details). Some microorganisms detected in this study have also been reported previously, such as \u003cem\u003eBartonella melophagi\u003c/em\u003e, \u003cem\u003eBa. schoenbuchensis, Arsenophonus endosymbiont\u003c/em\u003e, \u003cem\u003eWolbachia endosymbiont\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eNovosphingobium\u003c/em\u003e sp., and so on.\u003c/p\u003e \u003cp\u003eSymbiotic microorganisms may grow in the gut, body cavity, or cells of insects, potentially causing either positive effects, negative effects, reciprocal effects, or no apparent effect on the host [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The microbial population composition in insects can vary depending on diet, developmental stage, season, and geography [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The most abundant microbial genera detected in the study were \u003cem\u003eBartonella\u003c/em\u003e, \u003cem\u003eArsenophonus\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eTrypanosoma\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBartonella\u003c/em\u003e is a gram-negative bacterium transmitted to humans through blood-sucking arthropod vectors, or contact with contaminated animal feces, or on being scratched by infected animals [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Some studies have reported \u003cem\u003eBartonella\u003c/em\u003e sp. with a high rate of infection in \u003cem\u003eM. ovinus\u003c/em\u003e samples [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The last common ancestor of \u003cem\u003eBartonella\u003c/em\u003e was a likely amino acid and cofactor self-reliant gut symbiont that recycles nitrogenous waste products from its insect host; these symbionts can adapt to blood-sucking insects, but may not necessarily adapt to mammalian hosts, causing only opportunistic infections [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We detected \u003cem\u003eBartonella melophagi\u003c/em\u003e, \u003cem\u003eBa. schoenbuchensis, Ba. bovis\u003c/em\u003e, \u003cem\u003eBa. henselae\u003c/em\u003e, \u003cem\u003eBa. quintana\u003c/em\u003e, and so on in our samples. Of these, \u003cem\u003eBa. Henselae\u003c/em\u003e and \u003cem\u003eBa. quintana\u003c/em\u003e cause two serious diseases, cat scratch disease, and trench fever, respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e are considered insect symbionts that on the one hand, help their hosts to resist viruses and insecticides, and also aid in addressing some of the nutritional needs of their hosts [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], on the other hand, enhance their transmission, induce feminization, male-specific killing, and parthenogenesis of insect hosts [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The relative abundance of \u003cem\u003eWolbachia\u003c/em\u003e was as high as 67.8% in AT-5 pupae samples, much higher than that in adult \u003cem\u003eM. ovinus\u003c/em\u003e. As reported by studied by Duan et al., the abundance of \u003cem\u003eWolbachia\u003c/em\u003e in newly hatched and unfed \u003cem\u003eM. ovinus\u003c/em\u003e was about 29%, also higher than in the adult abundance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These values suggest that \u003cem\u003eWolbachia\u003c/em\u003e be supposed to have a greater effect on pupae or larvae than on adults.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eArsenophonus\u003c/em\u003e is a group of symbiont widely present in a several kinds of insects [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We detected \u003cem\u003eArsenophonus nasoniae\u003c/em\u003e, \u003cem\u003eAr. endosymbiont\u003c/em\u003e, \u003cem\u003eCandidatus\u003c/em\u003e Ar\u003cem\u003esenophonus Lipoptenae\u003c/em\u003e, and \u003cem\u003eArsenophonus\u003c/em\u003e sp. ENCA in our study. \u003cem\u003eAr. nasoniae\u003c/em\u003e infects people with symptoms of fever and pain [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Most of these symbionts are vertically transmitted from mother to offspring, while a small percent can also be transmitted horizontally [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. They affect insects and can also make people or animals sick. Therefore, these symbionts need to be researched further.\u003c/p\u003e \u003cp\u003eThe bacterium \u003cem\u003ePseudomonas\u003c/em\u003e is one of the most ubiquitous and diverse genera across the world, especially in a wide range of environments, objects, and organisms [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, we detected a variety of \u003cem\u003ePseudomonas\u003c/em\u003e sp., with its abundance in the AT-4 adult \u003cem\u003eM. ovinus\u003c/em\u003e samples being much higher than in other samples. This finding may be related to the suitable altitude and climate of the sample location for its growth. Test results included the opportunistic pathogen \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, capable of causing serious infections in human respiratory and urinary tracts [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We also detected a variety of bacterial pathogens, such as \u003cem\u003eBrucella abortus\u003c/em\u003e, \u003cem\u003eChlamydia trachomatis\u003c/em\u003e, \u003cem\u003eEnterobacter cloacae\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eYersinia pestis\u003c/em\u003e and so on, posing a serious threat to human or animal health.\u003c/p\u003e \u003cp\u003eProtozoa have a narrow host range and high specificity, and are thus also used as effective biocontrol agent of pest insects [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this study, a total of 45 parasitic protozoa were detected, except for \u003cem\u003eTrypanosoma\u003c/em\u003e, with a relatively high abundance across samples, while all other protozoa had a relatively low abundance. Until now, no study has reported on the assessment of the interrelationships between \u003cem\u003eM. ovinus\u003c/em\u003e and protozoa. The protozoa detected in this study are pathogenic, such as the highly pathogenic protozoa causing Chagas disease (\u003cem\u003eTrypanosoma cruzi\u003c/em\u003e) or sleeping sickness (\u003cem\u003eTrypanosoma brucei\u003c/em\u003e) in humans [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], T\u003cem\u003erichomonas vaginalis\u003c/em\u003e, which causes diseases of the human reproductive and urinary systems [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, which causes severe anemia and cerebral malaria [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, only five studies have reported the detection of viruses from \u003cem\u003eM. ovinus\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the current study, only the Orf virus and multiple phages were detected. The Orf virus is a DNA virus of genus \u003cem\u003eParapoxvirus\u003c/em\u003e, a highly contagious zoonotic disease causing a highly contagious vesiculo ulcerative pustular infection [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Phages present in \u003cem\u003eM. ovinus\u003c/em\u003e regulate the bacterial community. The absence of phages may cause an imbalance in the bacterial community in \u003cem\u003eM. ovinus\u003c/em\u003e and adversely affect its health. In this study only 11 phage species, including Wolbachia phage WO, Escherichia phage L AB-2017, and Staphylococcus phage VB-SauS-SA2, with very low abundance, were detected.\u003c/p\u003e \u003cp\u003eMicroorganisms that parasitize insect hosts can positively affect the host, such as providing nutrients or enhancing the fitness of the host while obtaining nutrients for themselves. The microorganisms may also be pathogenic to the insect host, reducing the fitness or causing its death. Therefore, studying microbial community diversity in \u003cem\u003eM. ovinus\u003c/em\u003e and determining their roles and functions will make it possible to efficiently investigate a way to manage pests. In this study, multiple pathogens were found in only one or two locations, suggesting that some pathogens may be endemic. During blood-sucking, \u003cem\u003eM. ovinus\u003c/em\u003e may lead to epidemics of these pathogens in sheep, which in turn may threaten public health safety or the stability of the sheep industry economy.\u003c/p\u003e \u003cp\u003eIn this study, a variety of microorganisms were detected, and the data on the microbial diversity of \u003cem\u003eM. ovinus\u003c/em\u003e in Xinjiang were enriched. However, the data on the microbial diversity of \u003cem\u003eM. ovinus\u003c/em\u003e in various places is still insignificant to. In conclusion, our study has implications regarding veterinary and public health safety. Based on the pathogen diversity, we need to assess their potential risk of \u003cem\u003eM. ovinus\u003c/em\u003e to animal husbandry and public health and control its infestations. The relationship, between microorganisms and \u003cem\u003eM. ovinus\u003c/em\u003e and its transmission and function can be used to develop methods for biological prevention and control of the pests.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, 32 microbial phyla were detected in sheep ticks in Xinjiang, China, with \u003cem\u003eProteobacteria\u003c/em\u003e being the dominant phylum. \u003cem\u003eBartonella\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eArsenophonus\u003c/em\u003e were the dominant genera among the 372 genera detected. After eliminating non-microbial species, 943 microorganisms, containing nearly 100 pathogens were detected. This is the first study to report most of the bacteria (e.g., \u003cem\u003eBartonella bovis\u003c/em\u003e and \u003cem\u003eArsenophonus nasoniae\u003c/em\u003e), fungi (e.g., \u003cem\u003eSaitoella complicata\u003c/em\u003e), viruses (e.g., Orf virus and Wolbachia phage WO), and protozoa (e.g., \u003cem\u003eTrypanosoma theileri\u003c/em\u003e and \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e) in \u003cem\u003eM. ovinus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnited States dollar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNR database\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-Redundant Protein Database.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank members of our laboratories for fruitful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants awarded by the National Natural Science Foundation of China (Granted No. 32160841 and 31960705), Key Laboratory of Tarim Animal Husbandry Science and Technology, Xinjiang Production \u0026amp; Construction Group (HS201802), and Tarim University Graduate Student Research Innovation Project (TDGRI202240).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw tags have been deposited in the Sequence Read Archive (SRA) from the NCBI under BioProject accession PRJNA1074431. The individual run files received accession numbers SRR27908141, SRR27908142, SRR27908143, SRR27908144 and SRR27908145. (A link to the BioProject and associated SRA metadata is ready to copy and share with reviewers via your publisher.\u003c/p\u003e\n\u003cp\u003ehttps://dataview.ncbi.nlm.nih.gov/object/PRJNA1074431?reviewer=sljjb8knean2cg8jidfsk7n3u1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJW and KH provided the research idea. XQ and KZ performed the collection and assembly of data. XZ and NX performed the experiments. QF and LS performed the data analysis and interpretation. KH wrote the manuscript. JW handled the critical revision of the article. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics approval was required as ectoparasite collection from the sheep was part of the routine medical procedure for rescued animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmall RW. A review of \u003cem\u003eMelophagus ovinus\u003c/em\u003e (L.), the sheep ked. 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Rev Med Virol. 2017;27.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Melophagus ovinus, Pupae, Metagenomics Sequencing, Microbial population, Xinjiang","lastPublishedDoi":"10.21203/rs.3.rs-3990667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3990667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMelophagus ovinus\u003c/em\u003e, a parasite on the body surface of sheep, directly attacks the host through biting and sucking blood and may also transmit pathogens in the process. There are currently only a few studies on the microbial composition of \u003cem\u003eM. ovinus\u003c/em\u003e, while there are no such studies on pupae.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eM. ovinus\u003c/em\u003e adults and pupae were collected from four regions in Xinjiang, China. DNA was extracted from the samples, amplified, and sequenced using the Illumina Novaseq 6000 System; finally, the sequencing data were analyzed using molecular biology software.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFrom all samples, a total of 32 phyla, comprising 372 genera and 1037 species, were detected. The highest microbial diversity was observed in Kuqa City (AT-2) and Qira County (AT-4). Pupae exhibited 40 unique microbial genera (AT-5) but did not have the highest microbial diversity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eProteobacteria was the dominant phylum in all samples. The dominant genera included \u003cem\u003eBartonella\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eArsenophonus\u003c/em\u003e. This is the first study to report most of the bacteria (e.g., \u003cem\u003eBartonella bovis\u003c/em\u003e and \u003cem\u003eArsenophonus nasoniae\u003c/em\u003e), fungi (e.g., \u003cem\u003eSaitoella complicata\u003c/em\u003e), viruses (e.g., Orf virus and Wolbachia phage WO), and protozoa (e.g., \u003cem\u003eTrypanosoma theileri\u003c/em\u003e and \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e) in \u003cem\u003eM. ovinus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"First metagenomic sequencing for the analysis of microbial community population of Melophagus ovinus and pupae in Xinjiang, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 12:11:35","doi":"10.21203/rs.3.rs-3990667/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"712437a7-6886-46f6-855c-1ca5b14724e4","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-06T16:41:20+00:00","versionOfRecord":{"articleIdentity":"rs-3990667","link":"https://doi.org/10.3389/fvets.2024.1462772","journal":{"identity":"frontiers-in-veterinary-science","isVorOnly":true,"title":"Frontiers in Veterinary Science"},"publishedOn":"2024-12-04 00:00:00","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-02-29 12:11:35","video":"","vorDoi":"10.3389/fvets.2024.1462772","vorDoiUrl":"https://doi.org/10.3389/fvets.2024.1462772","workflowStages":[]},"version":"v1","identity":"rs-3990667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3990667","identity":"rs-3990667","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}