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Micropathogen community identification in ticks (Acari: Ixodidae) using third-generation sequencing.

ABSTRACT: Ticks are important vectors that facilitate the transmission of a broad range of micropathogens to vertebrates, including humans. Because of their role in disease transmission, it has become increasingly important to identify and characterize the micropathogen profiles of tick populations. The objective of the present study was to survey the micropathogens of ticks by third-generation metagenomic sequencing using the PacBio Sequel platform. Approximately 46.481 Gbp of raw micropathogen sequence data were obtained from samples from four different regions of Heilongjiang Province, China. The clean consensus sequences were compared with host sequences and filtered at 90% similarity. Most of the identified genomes represent previously unsequenced strains. The draft genomes contain an average of 397,746 proteins predicted to be associated with micropathogens, over 30% of which do not have an adequate match in public databases. In these data, Anaplasma phagocytophilum and Coxiella burnetii were detected in all samples, while Borrelia burgdorferi was detected only in Ixodes persulcatus ticks from G1 samples. Viruses are a key component of micropathogen populations. In the present study, Simian foamy virus, Pustyn virus and Crimean-Congo haemorrhagic fever orthonairovirus were detected in different samples, and more than 10-30% of the viral community in all samples comprised unknown viruses. Deep metagenomic shotgun sequencing has emerged as a powerful tool to investigate the composition and function of complex microbial communities. Thus, our dataset substantially improves the coverage of tick micropathogen genomes in public databases and represents a valuable resource for micropathogen discovery and for studies of tick-borne diseases.

SUBMITTER: Luo J

PROVIDER: S-EPMC8253887 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MicroRNAs act as mRNA post-transcriptional regulators, playing important roles in cell differentiation, transcriptional regulation, growth, and development. In this study, microRNA expression profiles of Hyalomma anatolicum anatolicum ticks at different developmental stages were detected by high-throughput sequencing and functionally assessed. In total, 2,585,169, 1,252,678, 1,558,217, and 1,155,283 unique reads were obtained from eggs, larvae, nymphs, and adults, respectively, with 42, 46, 45, and 41 conserved microRNAs in these stages, respectively. Using eggs as a control, 48, 43, and 39 microRNAs were upregulated, and 3, 10, and 9 were downregulated in larvae, nymphs, and adults, respectively. MicroRNA-1 (miR-1) was expressed in high abundance throughout Ha. anatolicum development, with an average of nearly one million transcripts, and it is highly conserved among tick species. Quantitative real-time PCR (qPCR) showed that miR-1 expression gradually increased with tick development, reaching the highest level at engorgement. Differential tissue expression was detected, with significantly higher levels in the salivary glands and epidermis than in the midgut. Inhibition assays showed no significant change in body weight or spawning time or amount between experimental and control groups, but there was a significant difference (p < 0.01) in engorgement time. With miR-1 inhibition, ticks displayed obvious deformities during later development. To more fully explain the microRNA mechanism of action, the miR-1 cluster was analyzed according to the target gene; members that jointly act on Hsp60 include miR-5, miR-994, miR-969, and miR-1011. Therefore, microRNAs are critical for normal tick development, and the primary structure of the mature sequence of miR-1 is highly conserved. Nonetheless, different developmental stages and tissues show different expression patterns, with a certain role in prolonging feeding. miR-1, together with other cluster members, regulates mRNA function and may be used as a molecular marker for species origin, evolution analysis, and internal reference gene selection.

Project description:BACKGROUND:The goal of this study was to reassess the taxonomic status of A. maculatum, A. triste and A. tigrinum by phylogenetic analysis of five molecular markers [four mitochondrial: 12S rDNA, 16S rDNA, the control region (DL) and cytochrome c oxidase 1 (cox1), and one nuclear: ribosomal intergenic transcribed spacer 2 (ITS2)]. In addition, the phenotypic diversity of adult ticks identified as A. maculatum and A. triste from geographically distinct populations was thoroughly re-examined. RESULTS:Microscopic examination identified four putative morphotypes distinguishable by disjunct geographical ranges, but very scant fixed characters. Analysis of the separated mitochondrial datasets mostly resulted in conflicting tree topologies. Nuclear gene sequences were almost identical throughout the geographical ranges of the two species, suggesting a very recent, almost explosive radiation of the terminal operational taxonomic units. Analysis of concatenated molecular datasets was more informative and indicated that, although genetically very close to the A. maculatum - A. triste lineage, A. tigrinum was a monophyletic separate entity. Within the A. maculatum - A. triste cluster, three main clades were supported. The two morphotypes, corresponding to the western North American and eastern North American populations, consistently grouped in a single monophyletic clade with many shared mitochondrial sequences among ticks of the two areas. Ticks from the two remaining morphotypes, south-eastern South America and Peruvian, corresponded to two distinct clades. CONCLUSIONS:Given the paucity of morphological characters, the minimal genetic distance separating morphotypes, and more importantly the fact that two morphotypes are genetically indistinguishable, our data suggest that A. maculatum and A. triste should be synonymized and that morphological differences merely reflect very recent local adaptation to distinct environments in taxa that might be undergoing the first steps of speciation but have yet to complete lineage sorting. Nonetheless, future investigations using more sensitive nuclear markers and/or crossbreeding experiments might reveal the occurrence of very rapid speciation events in this group of taxa. Tentative node dating revealed that the A. tigrinum and A. maculatum - A. triste clades split about 2 Mya, while the A. maculatum - A.triste cluster radiated no earlier than 700,000 years ago.

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Micropathogen community identification in ticks (Acari: Ixodidae) using third-generation sequencing.

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