RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

Ticks (Acari: Ixodida) are blood-sucking arthropods globally recognized as vectors of numerous diseases. They are primarily responsible for the transmission of various pathogens, including viruses, rickettsiae, and blood parasites of animals. Ticks are second to mosquitoes in terms of disease transmission to humans. The continuous emergence of tick-borne diseases and acaricide resistance of ticks necessitates the development of new and more effective control agents and strategies; therefore, understanding of different aspects of tick biology and their interaction with pathogens is very crucial in developing effective control strategies. RNA interference (RNAi) has been widely used in the area of tick research as a versatile reverse genetic tool to elucidate the functions of various tick proteins. During the past decade, numerous studies on ticks utilized RNAi to evaluate potentially key tick proteins involved in blood feeding, reproduction, evasion of host immune response, interaction with pathogens, and pathogen transmission that may be targeted for tick and pathogen control. This chapter reviewed the application of RNAi in tick research over the past decade, focusing on the impact of this technique in the advancement of knowledge on tick and pathogen biology

Longistatin, a Plasminogen Activator, Is Key to the Availability of Blood-Meals for Ixodid Ticks

Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5−10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca++-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, β and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks

A Continuing Exploration of Tick–Virus Interactions Using Various Experimental Viral Infections of Hard Ticks

To fully unravel the ixodid ticks’ role as vectors of viral pathogens, their susceptibility to new control measures, and their ability to develop acaricide resistance, acclimatization of ticks under laboratory conditions is greatly needed. However, the unique and complicated feeding behavior of these ticks compared to that of other hematophagous arthropods requires efficient and effective techniques to infect them with tick-borne viruses (TBVs). In addition, relatively expensive maintenance of animals for blood feeding and associated concerns about animal welfare critically limit our understanding of TBVs. This mini review aims to summarize the current knowledge about the artificial infection of hard ticks with viral pathogens, which is currently used to elucidate virus transmission and vector competence and to discover immune modulators related to tick–virus interactions. This review will also present the advantages and limitations of the current techniques for tick infection. Fortunately, new artificial techniques arise, and the limitations of current protocols are greatly reduced as researchers continuously improve, streamline, and standardize the laboratory procedures to lower cost and produce better adoptability. In summary, convenient and low-cost techniques to study the interactions between ticks and TBVs provide a great opportunity to identify new targets for the future control of TBVs

HlSRB, a Class B Scavenger Receptor, Is Key to the Granulocyte-Mediated Microbial Phagocytosis in Ticks

Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods

Scavenger Receptor Mediates Systemic RNA Interference in Ticks

RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks

Need for enforcement of ethicolegal education – an analysis of the survey of postgraduate clinical trainees

BACKGROUND: The number of medical lawsuits in Japan was between 14 and 21 each year before 1998, but increased to 24 to 35 per year after 1999. There were 210 lawsuits during this 10-year period. There is a need for skills and knowledge related to ethics, which is as fundamental to the practice of medicine as basic sciences or clinical skills. in Japan education in ethics is relatively rare and its importance is not yet recognized. Establishing ethics education using legal precedents, which has already been achieved in Western countries, will be a very important issue in Japan. In the present study, a questionnaire survey was conducted among graduate intern doctors, in order to investigate whether ethics education using precedents might have a positive effect in Japan. METHODS: In 2002, a questionnaire survey entitled Physicians' Clinical Ethics was carried out in a compulsory orientation lecture given to trainees before they started clinical practice in our hospital. The attendees at this lecture were trainees who came from colleges in various districts of Japan. During the lecture, 102 questionnaires were distributed, completed by attendees and collected. The recovery rate was 100%. The questionnaire consisted of 22 questions (in three categories), of which 20 were answered by multiple choices, and the other two were answered by description. The time required to complete the questionnaire was about 10 minutes. RESULTS: The recovered questionnaires were analyzed using statistical analysis software (SPSS for Windows, Release 10.07J-1/June/2000), in addition to simple statistical analysis. answers using multiple choices for the 20 questions in the questionnaire were input into SPSS. The principal component analysis was performed for each question. As a result, the item that came to the fore was "legal precedent". Since many intern doctors were interested in understanding laws and precedents, learning about ethical considerations through education using precedents might better meet with their needs and interests. CONCLUSION: We applied a new method in which the results of principal component analysis and frequencies of answers to other questions were combined. From this we deduced that the precedent education used in Western countries was useful to help doctors acquire ethical sensitivity and was not against their will. A relationship was found between reading precedents and the influence of lawsuits, and it was thought that student participation-type precedent education would be useful for doctors in order to acquire ethical sensitivity

LKR/SDH Plays Important Roles throughout the Tick Life Cycle Including a Long Starvation Period

BACKGROUND:Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE:Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods