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

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

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

Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli olenev 1929 infesting Mongolian horses, and their amplification in egg and larval progenies

Babesia equi (EMA-1) and Babesia caballi (BC48) gene fragments were amplified by polymerase chain reaction (PCR), in blood samples, and partially fed-females and egg and larval progenies of Dermacentor nuttalli, collected from horses in Altanbulag, Tuv Province, Mongolia. While Babesia parasite DNA was detected in some horse blood samples during the first PCR, all positive cases in partially fed-female ticks, eggs and larvae were confirmed by nested PCR. Present study reinforces earlier similar findings in unfed D. nuttalli ticks collected from an open space vegetation in Bayanonjuul, Tuv Province in Central Mongolia, pointing to the most likely important role of D. nuttalli in the transmission of equine babesiosis in Mongolia. The detection of parasite DNA in eggs and larval progenies is likewise suggestive of transovarial parasite transmission in this tick species

Babesial Vector Tick Defensin against Babesia sp. Parasites▿ †

Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms

Silencing of HlSRB, HlVgR, and HlVg-1 genes and proteins in the whole body of <i>H. longicornis.</i>

<p>Individually or in combination of <i>HlSRB</i>, <i>HlVgR</i>, <i>HlVg-1</i>, and <i>luc</i> dsRNA(s) were injected into <i>H. longicornis</i> adult ticks. The injected ticks were left for 12 hours at 25°C and infested on the rabbits for four days and then ticks samples were collected for RNA extraction and the preparation of ticks protein lysates in each group. The name of each dsRNA group is indicated above. RT-PCR analysis (A). RT-PCR analysis was performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028407#pone-0028407-g001" target="_blank">Fig. 1</a>. (A). Western blot analysis (B). Tick lysates were subjected to SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The membrane was probed with mouse anti-rHlSRB, anti-rHlVgR, or anti-rHlVg-1 sera; mouse anti-actin serum was used as a control. The name of each dsRNA group is the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028407#pone-0028407-g001" target="_blank">Fig. 1</a>.</p

Expression profiles of HlSRB, HlVg-1, and HlVgR genes and proteins in different tissues of <i>H. longicornis</i>.

<p>Individually or in combination of <i>HlSRB</i>, <i>HlVg-1</i>, <i>HlVgR</i>, and <i>luc</i> dsRNA(s) were injected into <i>H. longicornis</i> adult ticks. The midguts and ovaries of dsRNA-injected ticks at 4 days of feeding were dissected out in 0.1% diethylpyrocarbonate-treated 1 × PBS (-) under a microscope. The name of each dsRNA group is indicated above. RT-PCR analysis and Western blot analysis were conducted using the midguts (A and B) and ovaries (C and D).</p

<b>Table 2.</b> Phenotypic changes of ticks injected with a single or a combination of different dsRNA(s).

a<p>Sixteen ticks were collected from the host for the subsequent experiments at 4 days after attachment.</p>b<p>These ratios show the fecundity of engorged females. Values are the means of ±SD.</p>c<p>These mortality rates show the percentages of number of dead ticks 20 days after drop-off to the total number of engorged ticks per treatment.</p>d<p>Hatchings from eggs to larvae were examined at 25°C in an incubator for 60 days.</p><p>*P<0.05, luc dsRNA-injected group vs. HlSRB-, HlVgR-, HlVg-1-, HlSRB/HlVgR-, HlSRB/HlVg-1-, HlSRB/luc-, HlVg-1/HlVgR-, HlVgR/HlSRB-, HlVg-1/HlSRB-, luc/HlSRB-, and HlVgR/HlVg-1 dsRNA-injected groups.</p><p>**<i>P</i><0.05, <i>luc</i> dsRNA-injected group vs. <i>HlSRB-</i>, <i>HlVgR-</i>, <i>HlVg-1-</i>, <i>HlSRB/HlVgR-</i>, <i>HlSRB/HlVg-1-</i>, <i>HlSRB/luc-</i>, and <i>luc/HlSRB</i> dsRNA-injected groups.</p