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A microRNA profile of saliva and role of miR-375 in Haemaphysalis longicornis (Ixodida: Ixodidae)

ABSTRACT: 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, and it contained 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. The 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. 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 miRNAs 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.

ORGANISM(S): Haemaphysalis longicornis

PROVIDER: GSE123407 | GEO | 2019/01/31

REPOSITORIES: GEO

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Project description:Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in salivary glands of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.

Project description:Anti-tick vaccines have proved to be an effective and sustainable method for the control of tick infestations and tick-borne diseases with clear advantages over the application of chemical acaricides (Šmit and Postma, 2016; de la Fuente, 2018; Ndawula and Tabor, 2020). Moreover, the efficacy of acaricide application against Ornithodoros ticks is seriously limited owing to their endophilic/nidicolous life style, which make these ticks less accessible to the chemical acaricides (Astigaraga et al., 1995). Success in tick vaccine development is largely dependent on identification of new and highly protective tick antigens. Searching of new candidate protective antigens is currently being approached among tick molecules that play important biological functions at the tick-host interface, and more precisely among the salivary and intestinal proteins involved in biological processes specifically evolved by ticks to adapt to haematophagy (de la Fuente et al., 2016; Oleaga et al., 2021; Pérez-Sánchez et al., 2021). Accordingly, next-generation sequencing (NGS) and high-throughput proteomics technologies are been used to explore the transcriptome and proteome of the salivary glands/saliva and midguts of an increasing number of tick species and obtain the corresponding sialomes and mialomes (Chmelař et al., 2016; Almeida-Martins et al., 2020; Mans et al., 2020; Oleaga et al., 2021). These studies have identified a wealth of tick molecules related to tick haematophagy, tick-host interplay and pathogen transmission, which can then be scrutinized and filtered in vaccinomics pipelines for selecting candidate protective antigens (Chmelař et al., 2016; Maruyama et al., 2017; Antunes et al., 2018; de la Fuente et al., 2018; Ren et al., 2019; Couto et al., 2021). Similarly, we were also interested in characterizing the O. erraticus sialome. As far as O. erraticus saliva must contain all the bioactive molecules that the tick need to successfully feed, decoding its composition will lead to the discovery of new antigen targets for developing vaccines for the control and prevention of O. erraticus infestations and the diseases it transmits. Accordingly, the objective of the present work was to obtain the proteome of the saliva of O. erraticus adult ticks. For this, we have used a proteomics informed by transcriptomics approach to analyse female and male saliva separately using two different mass spectrometry approaches: liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode, and Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH MS). SWATH MS is a specific variant of data-independent acquisition (DIA) methods that combines deep proteome coverage capabilities with quantitative consistency and accuracy (Ludwig et al., 2018). Here we reported the identification of 387 non-redundant proteins in the saliva of O. erraticus adult ticks as well as a qualitative and quantitative comparison of the saliva protein composition between both sexes. The integration of O. erraticus sialoproteomic and sialotranscriptomic datasets facilitate a better understanding of the physiology of feeding in O. erraticus and will drive the discovery of new and more effective antigen targets for development of anti-tick vaccines.

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A microRNA profile of saliva and role of miR-375 in Haemaphysalis longicornis (Ixodida: Ixodidae)

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