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 Arsenophonus 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], Trichomonas 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.