Ticks are found in almost every region of the world and are second only to mosquitoes in their public health and veterinary importance 1. Ticks transmit the greatest variety of pathogens to humans and veterinary species of any arthropod vector 2, including the agents causing Anaplasmosis, Cowdriosis, East Coast Fever, Babesiosis, Lyme borreliosis, tick-borne relapsing fever, ehrlichiosis, Rocky Mountain spotted fever, Boutonneuse fever, Queensland tick typhus, Q fever and numerous arboviruses 3. Ticks in the Ixodes persulcatus complex, including the blacklegged tick, Ixodes scapularis, are particularly important vectors of human disease-causing agents; I. scapularis ticks can harbor multiple pathogens including the agents of Lyme disease, Borrelia burgdorferi 4, human anaplasmosis, Anaplasma phagocytophilum 5, human babesiosis, Babesia microti 6 and an encephalitis-like virus 7. Ixodes ticks have a life cycle involving egg, larval, nymphal and adult stages. Infected nymphal ticks play the most important role in transmitting disease to humans 8.

Sequencing of the I. scapularis genome, along with development of new investigative tools such as expressed sequence tags, microarrays, and RNA interference offer new alternatives for researching tick and tick-borne disease control 91011121314. One very promising method for generating targeted down-regulation of gene expression in a wide range of organisms is RNA interference (RNAi). RNAi is the process by which dsRNA inhibits accumulation of homologous transcripts from cognate genes, thus providing a powerful alternative to more traditional immunization and genetic techniques 15. RNAi also has evolved into a powerful tool for probing gene function in Drosophila, Tribolium, Caenorhabditis elegans and mice 1617. Delivery of dsRNA or siRNA into a cell triggers abrogation of the target mRNA. Previous experiments showed the feasibility of using RNAi to abrogate expression of gene transcripts in salivary glands of the ticks, Amblyomma americanum and Ixodes scapularis 18121314181516171819. Recently, Hatta et al., 21 demonstrated the RNA interference of cystosolic leucine aminopeptidase not only in the salivary glands, but also in the midgut, ovary and epidermis of Haemaphysalis longicornis.

A number of animal cells have been shown to naturally take up exogenous dsRNA and use it to initiate RNAi silencing 2223. In some organisms, such as Drosophila, certain cells efficiently uptake dsRNA but seem to be unable to transmit this dsRNA to other cells in the fly body 24. Organisms like C. elegans and juvenile grasshoppers can both take up dsRNA and spread it systemically to elicit an RNAi response throughout the entire organism 2526. The typical RNAi response occurs in two phases. In the first phase, double-stranded RNAs are cleaved into double stranded fragments called siRNAs by the RNase III enzyme Dicer 272829. In the second phase, these siRNAs are conveyed into a large ribonucleoprotein complex known as the RISC [RNA-induced silencing complex] where they act as guides, base-pairing to complementary mRNAs and triggering RISC-mediated mRNA cleavage 27293031321633 and destruction 343536. The mechanisms of uptake, spreading and processing of dsRNA are poorly understood. Labeled siRNA or dsRNA has been used in tracking of these molecules in mammalian cell lines and in schistosomes mansoni 373839. Here, we extend the scope of the RNAi technique in I. scapularis by describing the uptake and spread of dsRNA in tick synganglia [brain]. To test protocols for gene silencing in tick synganglia, we focused on β-Actin and Na⁺-K⁺-ATPases, genes that plays a conserved role in all tick tissues. We show that gene expression in the synganglia of female adult ticks, as exemplified by the genes coding for the β-Actin and Na⁺-K⁺-ATPase, can be specifically inhibited by microinjection of dsRNA into unfed ticks. In the future, this may make possible the targeting of genes encoding specific neuropeptides expressed only in the synganglia and that play vital roles in tick biology.

One of the major problems in generating dsRNA-mediated gene silencing is delivery of the dsRNA to the target site. Often, distribution of injected dsRNA cannot be visualized, and phenotypes are analyzed in all injected ticks under the assumption that dsRNA are uniformly distributed. To determine whether our dsRNAs became incorporated in tick synganglia and other tissues, we used Cy3 labeled Actin dsRNA. Use of Cy3 labeled dsRNAs permitted monitoring of the uptake of these molecules in tick tissues. Although injections were performed in the tick hemocoel, labeled dsRNA diffused throughout the entire tick (Fig. 1C and 1D). We also observed dotted localization of labeled dsRNA actin in whole adult female ticks (Fig. 1C). This ubiquitous distribution of labeled dsRNAs suggests that dsRNA is rapidly taken up and efficiently distributed throughout the cells in all tick tissues including neuronal cells (Fig. 1D). The accumulation of dotted staining (Fig. 1C) could be a hint that excess labeled dsRNAs can be secreted out by the feeding ticks. The fluorescence also was detected in the tick synganglia at various intensity levels (Fig. 2) indicating that molecules of dsRNA injected into adult female ticks can be up-taken by the synganglia where they would be expected to disrupt target gene expression. The result suggests that depletion of gene expression by dsRNA correlated with localization of fluorescently labeled dsRNA in the synganglia. To further assess the intensity and spread of labeled dsRNA, we examined the tick tissues for the presence of dsRNAs daily after injection. In the first 24 hrs, we observed the uptake of labeled dsRNA in synganglia cells (Fig 2E–F). This result suggests that dsRNA is efficiently up-taken not only in the salivary glands and the midgut tissues but also in synganglia.

Incubating dissected tick synganglia in vitro for 6 hrs with specific β-Actin- and Na⁺-K⁺-ATPase-targeted dsRNA demonstrated complete disruption of the specific gene transcript (Fig. 3A–B). The level of irrelevant genes Cyclophilin A and G remained unchanged in both the dsRNA injected and control groups, respectively (Fig. 3A–B). Unfed adult female ticks injected with either β-Actin or Na⁺-K⁺-ATPases dsRNA and then allowed to partially blood feed on a rabbit also showed reduction in endogenous gene transcripts (Fig 4A–B). Interestingly, a complete to partial decrease in target transcript levels was observed in synganglia dissected from replete ticks (data not shown).

A complete gene silencing also was observed in salivary glands (Fig 5A–B) as compared to control groups. We also observed suppression of β-Actin and Na⁺-K⁺-ATPase gene transcript in tissues like midguts, malpighian tubules and ovaries (data not Shown). β-Actin dsRNA-injected ticks blood fed on hosts and attained an average weight of ~50 mg compared to an average weight of ~190 mg in mock-injected controls (Fig 6D). RNAi-mediated reduction of β-Actin gene expression in ticks affected their ability to oviposit normally; β-Actin suppressed ticks laid small egg masses as compared to control groups (Fig. 6A–C). Na⁺-K⁺-ATPases dsRNA injected ticks blood fed on hosts attained an average weight of 20 mg compared to an average weight of partially fed control ticks of 100 mg (Fig. 7D). Na⁺-K⁺-ATPases gene silencing in tick tissues affected the ability of gene silenced ticks to oviposit or feed successfully (Fig. 7A–C).

Our results establish for the first time that dsRNA injections into adult female ticks can be used to trigger RNAi and cause the consequent depletion of respective gene products from tick organs such as synganglia (the CNS). The two gene products we targeted in the tick synganglia were endogenous β-Actin and Na⁺-K⁺-ATPases mRNA. Our present findings, that RNAi can be used for silencing genes in tick synganglia will help develop strategies for understanding the functional role of newly identified genes. RNAi is becoming the most widely used experimental technique for studying gene function in ticks. Previous experiments demonstrated systemic RNAi in ticks after injecting dsRNA into unfed ticks, and the resulting gene silencing could be detected in multiple tick tissues 14. These studies extend gene silencing to tissues like the synganglia. In the past, gene silencing in tick synganglia has been completely ignored largely because of the small size of the tissue and consequent challenges in validating an effect. Moreover, until recently, the number of tick synganglia target genes has been limited. The phenomenon of RNAi silencing is widely conserved among all higher eukaryotes, and exploiting this process is becoming increasingly important as an experimental tool, as well as for therapeutic applications. Although most cells possess the basic RNAi core machinery, some cell types have the intriguing ability to naturally take up exogenous dsRNA and use it to initiate RNAi silencing 2223404142. Furthermore, some organisms, such as plants, C. elegans and planaria (Giardia tigrina) are able to transmit the RNA silencing signaling from cell to cell, resulting in systemic spread of the RNAi response 43444546.

It has been shown in ixodid ticks that the Na⁺-K⁺-ATPases are sensitive to Ouabain [Na⁺ K⁺ pump blocker], and that the volume of saliva secreted is dependent on an active Na⁺-K⁺-ATPase machinery. Extrusion of Na⁺ by endogenous Na⁺-K⁺-ATPases enables animal cells to control their water content osmotically. Without active Na⁺-K⁺-ATPases, cells lacking cell walls (like animal cells) would swell and burst. In neuronal cells, the electrochemical potential gradient generated by Na⁺-K⁺-ATPase activity is responsible for the electrical excitability of nerve cells, while in other cells (such as intestinal epithelium and erythrocytes) it provides the free energy necessary for active transport of glucose and amino acids. Inactivating Na⁺-K⁺-ATPases affects exocytosis 4748. In ixodid ticks, correlation between fluid secretion and Na⁺-K⁺-ATPases activity is highest in the salivary glands of mated, rapidly feeding females. Inactivating Na⁺-K⁺-ATPases does not affect protein secretion, but rather the volume of saliva secreted 49, implying that Na⁺-K⁺-ATPases are involved in maintaining cellular osmolarity in tick salivary glands.

The amount of both Na⁺-K⁺-ATPases and β-Actin mRNA in the adult tick synganglia is considerable, and our results suggest that RNAi can similarly be used to suppress other abundantly expressed genes in tick synganglia. It should be noted that the observed high efficiency of the dsRNA injection technique for inhibiting gene expression may be due to the structure and function of the tick synganglia. When dsRNA is injected into the tick hemocoel, the synganglia tissue is readily exposed. Similar systemic results have been noted in other arthropods with open circulatory systems. In Drosophila melanogaster, Dzitoyeva et al. 50 demonstrated that intra-abdominal injections of homologous dsRNA efficiently silenced LacZ transgene expression in the fruit fly's gut as well as in their optic and antennal lobes, and that the method is potent in silencing endogenous GM06434 mRNA in the central nervous system. Even if the scope of this technique should turn out to be more restricted in ticks than in Drosophila, our results suggest that a number of important genes being expressed in the tick synganglia can be targeted this way.

Although it is commonly reported that RNAi is an evolutionarily conserved mechanism, molecular differences among several model systems are being discovered 51. Thus, cells of most invertebrates in which RNAi has been used successfully are able to take up dsRNA molecules by themselves, with the exception being Drosophila. The molecule responsible for dsRNA uptake and siRNA spreading is SID-1, a specific transmembrane protein 525354. Although Drosophila is susceptible to the effects of RNAi, they lack a sid-1 ortholog and do not exhibit the spreading characteristic seen more typically in other invertebrate systems 5354. Because of this, the amount of dsRNA needed for effective RNAi in Drosophila is 10,000 times higher than in controls expressing sid-1 ectopically.

We observed substantial RNAi suppression of two ubiquitous genes also present in tick synganglia obtained from unfed adults injected with dsRNA, and this method seems as though it will provide a means for disrupting specific gene function in the synganglia. With the number of (partially) sequenced tick genes steadily increasing in electronic databases (i.e. 1472 Expressed Sequence Tags of March 13, 2008 55, it is encouraging that we apparently will not need to wait for the development of sophisticated delivery techniques before starting advanced reverse genetics on tick synganglia.

RNAi is a method by which dsRNA can be introduced directly into ticks to effect significant suppression of specific gene expression, even in fully developed tick synganglion. In light of our results, we are confident that conservation of the systemic RNAi pathway in ticks paves the way for more comprehensive studies to molecularly dissect the important roles of neuropeptides in tick biology. The systemic RNAi pathway also may provide opportunities for developing species-specific, and hence, ecologically friendly tick control methods. In the future, availability of annotated genome sequence information for different tick species 95657 would pave the way for genome-wide RNAi applications addressing fundamental questions in tick neurophysiology, development and gene regulation.

Unless otherwise indicated, the protocols followed standard procedures 58, and all the experiments were performed at room temperature (25 ± 1°C). All materials were obtained from Sigma-Aldrich (St. Louis, MO, USA) except the water which was of 18 MΩ quality, produced by a MilliQ apparatus (Millpore, Bedford, MA, USA).

RNAi-RNA interference, dsRNA-double stranded RNA, TBD-tick borne diseases, PCR-polymerase reaction, Na K ATPase-Sodium Potassium ATPase.

SK conceived the study, carried out the RNAi studies and drafted the manuscript. BK carried out the RNA extraction and RT-PCR. ET performed all Dicer expression experiments. TNM participated in the tick collection and micro-dissections, statistical analysis and helped draft the manuscript. All authors read and approved the final manuscript.