Tick saliva contains many bioactive molecules that are involved in attachment to the host, blood-feeding and transmission of pathogens. MicroRNAs (miRNAs) are a class of short non-coding RNAs with a length of 19–24 nucleotides. They act as regulators of gene expression by binding to their target mRNA at the post-transcriptional level and control a variety of cellular functions, including regulation of growth, metabolism and development. The detection and characterizations of miRNAs from tick saliva may help explain the molecular mechanisms involved in the interaction between ticks, pathogens and hosts. They may also contribute to the discovery of vaccines, which can control ticks and the pathogens they transmit. An RNA library was generated from the saliva of fed adult Haemaphysalis longicornis ticks, containing 17.4 million clean reads of 18–30 nucleotides. Overall, 319 known miRNAs and 1 novel miRNA were found. The 10 most abundantly expressed miRNAs present in tick saliva were miR-100_2, miR-315, miR-184_1, miR-100-5p_2, miR-5307, miR-184-3p_3, Let-7-5p_6, miR-71_5, miR-1-3p_6 and miR-10-5p_2. miR-375, one of the abundantly expressed, was subjected to quantitative real-time PCR analysis (qRT-PCR) in various tick developmental stages, as well as in different tissues isolated from adult ticks. The expression of miR-375 in different tick development stages was highest in unfed nymphs and lowest in the egg stage. In the tissues of adult ticks, miR-375 was most highly expressed in the salivary gland. To investigate the possible role of miR-375, Ant-375 was used to inhibit the miR-375. The treated group (Ant-375) had a reduced number of eggs (t(10) = 2.652, P = 0.0242), eggs that were partially desiccated, and reduced egg hatchability (t(10) = 2.272, P = 0.044) compared to Ms-Ant and the non-injected control. This is the first study to investigate the miRNA profile in tick saliva and the role of miR-375 in H. longicornis. The identification and characterization of miRNA in tick saliva may help to reveal the molecular mechanisms of interactions among ticks, pathogens and hosts, and suggest new vaccine strategies to control tick-borne diseases.

Ticks are considered the second most common pathogen vectors transmitting a broad range of vital human and veterinary viruses. From 2017 to 2018, 640 ticks were collected in eight different provinces in central and western China. Six species were detected, including H.longicornis, De.everestianus, Rh.microplus, Rh.turanicus, Rh.sanguineous, and Hy.asiaticum. Sixty-four viral metagenomic libraries were constructed on the MiSeq Illumina platform, resulting in 13.44 ​G (5.88 ​× ​10⁷) of 250-bp-end reads, in which 2,437,941 are viral reads. We found 27 nearly complete genome sequences, including 16 genome sequences encoding entire protein-coding regions (lack of 3′ or 5′ end non-coding regions) and complete viral genomes, distributed in the arboviral family (Chuviridae, Rhabdoviridae, Nairoviridae, Phenuiviridae, Flaviviridae, Iflaviridae) as well as Parvoviridae and Polyomaviridae that cause disease in mammals and even humans. In addition, 13 virus sequences found in Chuviridae, Nairoviridae, Flaviviridae, Iflaviridae, Hepeviridae, Parvoviridae, and Polyomaviridae were identified as belonging to a new virus species in the identified viral genera. Besides, an epidemiological survey shows a high prevalence (9.38% and 15.63%) of two viruses (Ovine Copiparvovirus and Bovine parvovirus 2) in the tick cohort.

Ticks, as blood-feeding arthropod vectors, have evolved their own unique mechanism to suppress host immune responses and evade immune defenses in order to complete blood-feeding. The immunoregulatory effect of tick bioactive molecules on hosts has been widely reported, and the cystatin family has been identified as one of the major immunomodulators. In previous studies, we obtained a novel tick salivary bioactive protein named RHcyst-1, which belongs to the type 1 cystatin family. Here, we demonstrated the effects of RHcyst-1 on the host immune response mainly on dendritic cell (DC) function. Understanding the function of tick-derived bioactive molecule may help to clarify the mechanisms of how ticks escape the host immune response and help to control ticks and tick-borne disease transmission. Bone marrow-derived DCs (BMDCs) were generated and induced by GM-CSF and IL-4 with or without RHcyst-1 addition. Flow cytometry was used to analyze the differentiation and maturation of BMDCs and T cell cytokine production. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure changes in expression within STAT and p38 MAPK signaling pathways. Flow cytometry analysis revealed that RHcyst-1 inhibited the differentiation of BMDCs, but had no effect on the maturation of BMDCs. T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNF-α, IFN-γ and IL-2 than the control group. Further analysis showed that the mRNA level and phosphorylation of p38, ERK and STAT were significantly changed after RHcyst-1 added to bone marrow monocytes during the differentiation stage. Our results suggest that RHcyst-1 is one of the major immunosuppressive proteins of BMDC function from blood-feeding ticks.