RNA interference for the study and genetic manipulation of ticks

Ticks are ectoparasites of wild and domestic animals, and humans. A more comprehensive understanding of tick function and the tick-pathogen interface is needed to formulate improved tick-control methods. RNA interference (RNAi) is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulations has been limited. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface, and the screening and characterization of tick protective antigens. This review considers the applications of RNAi to tick research and the potential of this technique for tick functional studies, and to elucidate the tick-pathogen and tick-host interface. It is probable that the knowledge gained from this experimental approach will contribute to development of vaccines to control tick infestations and the transmission of tick-borne pathogens.     

Single-chain Fv phage display propensity exhibits strong positive correlation with overall expression levels

Background

Progress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (Ixodes scapularis). However, proving that RNAi can work in nervous tissue has been problematic. Developing an ability to manipulate gene expression in the tick synganglia likely would accelerate understanding of tick neurobiology. Here, we assess gene silencing by RNA interference in the adult female black-legged tick synganglia.

Results

Tick β-Actin and Na⁺-K⁺-ATPase were chosen as targets because both genes express in all tick tissues including synganglia. This allowed us to deliver dsRNA in the unfed adult female ticks and follow a) uptake of dsRNA and b) gene disruption in synganglia. In vitro assays demonstrated total disruption of both tick β-Actin and Na⁺-K⁺-ATPase in the synganglia, salivary glands and midguts. When dsRNA was microinjected in unfed adult female ticks, nearly all exhibited target gene disruption in the synganglia once ticks were partially blood fed.

Conclusion

Abdominal injection of dsRNA into unfed adult female ticks appears to silence target gene expression even in the tick synganglia. The ability of dsRNA to cross the blood-brain barrier in ticks suggests that RNAi should prove to be a useful method for dissecting function of synganglia genes expressing specific neuropeptides in order to better assess their role in tick biology.     

Ticks (Acari: Ixodoidea) in China: Geographical distribution,host diversity,and specificity

Ticks are obligate blood‐sucking ectoparasites, which not only directly damage through bites but also transmit many pathogens. China has a high diversity of tick species, 125 species have been reported, including 111 hard tick and 14 soft tick species. Many of the ticks are important vectors of pathogens, resulting in zoonoses. The dynamics of ticks are affected by both the host and habitat environment. However, systematic studies on the geographical distribution, host diversity, and specificity of ticks are limited in China. To achieve this goal, the relevant available data were summarized and analyzed in this study. Ticks are distributed in all parts of China and Xinjiang has the most records of ticks. The distribution of ticks in adjacent areas is similar, indicating that the habitat environment affects their distribution. Most ticks are widely distributed, whereas some species are endemic to their distributed regions. Ticks are parasitic on mammals, birds, and reptiles, of which mammals are the main host species. Overall, most ticks parasitize different hosts, only a few ticks have strict host specificity, such as ticks that are specifically parasitic on reptiles and bats. In addition, environmental changes and control efforts also influence the dynamics of ticks. These results can better reveal tick biological traits and are valuable for tick control.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.     

Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks

Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods.     

Ticks (Acari: Ixodida) infesting humans in the provinces of Kelkit Valley, a Crimean-congo hemorrhagic fever endemic region in Turkey

Ticks are mandatory blood feeding ectoparasites leading transmission of various tick-borne pathogens to human and animals. Since 2002, thousands of human tick bites and numerous Crimean-Congo Hemorrhagic Fever cases have been reported in several provinces in the Kelkit Valley region in Turkey. Despite increased cases of tick bites and tick-borne diseases, no taxonomic information is available about the tick species infesting humans in the region. In the present study, a tick survey on humans was performed to determine the species composition of ticks infesting humans in several provinces of Kelkit Valley. In the survey, 1,460 ticks (721 males, 516 females and 223 nymphs) were collected from tick-infested humans. A total of 19 tick species have been found on humans in the region, including 7 Hyalomma, 2 Argas, 2 Haemaphysalis, 2 Ixodes, Dermacentor and 3 Rhipicephalus species. Infestation of Dermacentor reticulatus on humans was documented for the first time in Turkey.     

A review of the ticks (Acari: Ixodida) of Turkey: species diversity, hosts and geographical distribution

Ticks are important ectoparasites, causing a variety of serious infectious diseases in humans and domestic animals. There is very limited taxonomic information about the tick species of Turkey in the literature, even though Turkey has very suitable climate and vegetation for ticks. In the current study, species diversity, hosts and geographical distribution of the ticks present in Turkey are reviewed based on taxonomic data in the literature from 1915 to 2011 and our recent observations. The names of tick species are arranged according to the most recent check lists. The taxonomic records in the literature and our studies on actual tick samples indicated that the tick fauna of Turkey consists of 46 species; 38 species from Ixodidae and 8 species from Argasidae.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Application of RNA interference in tick salivary gland research.

Ticks are obligate ectoparasites that feed on a variety of hosts including mammals, birds and reptiles. Prolonged attachment on the host and an ability to transmit a wide variety of pathogens are the special features of tick feeding. Salivary glands are the major route for secretion of excess fluid, several proteins, and factors that counteract the host immune response and hence play a significant role in the success of tick feeding. RNA interference (RNAi) enables scientists to silence genes encoding proteins in an absolutely sequence specific manner at the mRNA level. This technique has already been successfully employed in analyzing roles of proteins of important functions or in assigning roles to several proteins of unknown functions in a variety of animals. In this review, we outline the process of RNAi and the applicability of RNAi in tick salivary gland research.     

A tick from a prehistoric Arizona coprolite

Ticks have never been reported in archaeological analyses. Here, we present the discovery of a tick from a coprolite excavated from Antelope Cave in extreme northwest Arizona. Dietary analysis indicates that the coprolite has a human origin. This archaeological occupation is associated with the Ancestral Pueblo culture (Anasazi). This discovery supports previous hypotheses that ticks were a potential source of disease and that ectoparasites were eaten by ancient people.     

Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.     

Species,developmental stage and infection with microbial pathogens of engorged ticks removed from dogs and questing ticks

Research into tick‐borne diseases implies vector sampling and the detection and identification of microbial pathogens. Ticks were collected simultaneously from dogs that had been exposed to tick bites and by flagging the ground in the area in which the dogs had been exposed. In total, 200 ticks were sampled, of which 104 came from dogs and 96 were collected by flagging. These ticks were subsequently examined for DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia spp. and Babesia canis. A mixed sample of adult ticks and nymphs of Ixodes ricinus (Ixodida: Ixodidae) and Haemaphysalis concinna (Ixodida: Ixodidae) was obtained by flagging. Female I. ricinus and adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks dominated the engorged ticks removed from dogs. Rickettsia spp. were detected in 17.0% of the examined ticks, A. phagocytophilum in 3.5%, B. canis in 1.5%, and B. burgdorferi s.l. in 16.0%. Ticks with multiple infections were found only among the flagging sample. The ticks removed from the dogs included 22 infected ticks, whereas the flagging sample included 44 infected ticks. The results showed that the method for collecting ticks influences the species composition of the sample and enables the detection of a different pattern of pathogens. Sampling strategies should be taken into consideration when interpreting studies on tick‐borne pathogens.