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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.
Small RNA–mediated gene silencing, or RNA interference (RNAi), was discovered recently when it was shown that double-stranded RNA (dsRNA), when injected into the nematode Caenorhabditis elegans, silenced the gene of absolute sequence similarity.1 Since then, RNAi has been effectively used to study and analyze the functions of several proteins in several species.2–4 Simplicity and specificity, the key features of RNAi, have made it a powerful tool for silencing and RNAi has largely replaced the conventional methods of gene silencing, which are more cumbersome and time consuming.
The inducer of RNAi is dsRNA.5 dsRNA is processed by an RNaseIII-type enzyme, namely Dicer, to form small inhibitory RNAs (siRNAs).6 siRNAs are double-stranded RNA molecules, 21–26 nt in length. siRNAs initiate RNAi by recruiting proteins to form a ribonucleoprotein complex called RISC (RNAi silencing complex).7 Although a protein designated as Argonaute8,9 and an RNA-directed RNA polymerase10 have been found to be part of the complex ribonucleoprotein, the identities of all the proteins involved are not yet fully understood. The siRNA then binds to mRNA of absolute sequence similarity and causes its degradation.
Micro RNAs (miRNAs) are also short interfering RNAs that have been shown to play a role in gene silencing.11 siRNAs are produced from the target gene itself; for example, a double-stranded virus, transposons, or transgene. miRNAs, on the other hand, are produced from self-complementary transcript regions within an organism that do not code for protein. Both siRNAs and miRNAs are produced by the action of Dicer, probably by two different homologs—Dicer1 and Dicer2.12 Once the small RNAs are produced, they become associated with proteins to form the RISC, as described above. siRISC binds to a target gene of absolute gene specificity (the same sequence as the dsRNA), whereas miRISC binds to target genes with partial sequence specificity and results in translational repression of that particular gene, rather than mRNA degradation as in the case of siRISC (Figure 11).
Ticks feed on a variety of hosts, including cattle, sheep, deer, dogs, and humans. Ticks are second only to mosquitoes in vectoring diseases, but are the foremost arthropod vector in the variety of pathogens they transmit. Ticks belong to the class Arachnids and subclass Acari. Parasitiformes is an order under the subclass Acari. Parasitiformes have a suborder termed as Ixodida. The suborder Ixodida can be further classified into two major families of ticks, the Argasidae (soft ticks) and the Ixodidae (hard ticks). Nuttalliellidae, a third family of ticks, also exists, but consists of just one species. Argasid ticks feed very rapidly and do not ingest blood meals as large as ixodid ticks. Ixodid ticks feed for prolonged periods on hosts and transmit several pathogens, such as the causative agents of diseases like Rocky Mountain spotted fever, Lyme disease, ehrlichiosis, and anaplasmosis. A typical lone star female tick increases from about 4 mg (unfed) to about 40 mg after 6 days of feeding. After 12 days of feeding, they weigh approximately 150 mg. Then they enter a fast feeding stage and engorge to repletion within 24–48 h, attaining a weight of approximately 600 mg (Figure 22).). Female lone star ticks will not continue to feed, and attain a weight greater than 35–50 mg in the absence of mating, which occurs on the host.13
Tick salivary glands (TSGs) are keys to the successful feeding of an ixodid tick on a host. Salivary glands are organs that grow and differentiate tremendously as the tick continues to feed and grow.14 The pair of acinous salivary glands is located in the anterior end of the tick (Figure 33).). The physiology, the variety of factors they secrete, and the importance of these factors in tick feeding have been reviewed in detail.15,16 One limiting factor in tick research is the lack of information available on processes, proteins, and other key secretions that govern tick feeding. There has been relatively little progress in the use of cutting-edge research methodologies in ticks due to the lack of a genetic system and genomic and proteomic information until recently. Expressed sequence tag (EST) projects have yielded much information on the sequences of several genes encoding proteins of significant function. Not unexpectedly, the EST projects have identified many genes encoding proteins of unknown functions.17,18 Further information is required to confirm their functional significance in tick feeding and growth.19 RNAi provides a comparatively easy, reliable, inexpensive, and powerful tool to analyze the functions of these proteins within tick salivary glands and whole ticks as well. In the following section, RNAi performed on several genes in the tick salivary glands and the potential it has in studying the tick system will be reviewed. Our purpose is to show how RNAi and its silencing effects can be used within tick salivary glands to gain information about tick salivary gland function. Similar studies could be performed in any primitive organism with lesser available information. This could result in great and novel insights into how these organisms grow and regulate complex processes, and how they are similar to or different from related species.
The long duration of tick feeding on the host has generated much interest in studying the factors that ticks secrete to counteract the host immunological and inflammatory responses.20,21 Histamine is a factor released by the host to the site of inflammation that hinders pathogen growth. Ticks secrete a protein that binds to the free histamine, histamine binding protein (HBP) at the feeding site, thereby presumably suppressing the effect of the inflammatory mediator.22,23 A differential gene-expression project with salivary glands from male lone star ticks identified a gene sequence that had significant homology to HBPs from other tick species at the amino acid level.24 The first report of RNAi applicability in tick research was published in 2003, when RNAi, using a dsRNA derived from this gene, was shown to affect HBP mRNA levels in female lone star tick salivary glands.25 In addition to the molecular data that showed the differences in the transcript levels of HBP in vitro, the authors were able to show that dsRNA, when injected into unfed ticks, was able to enter the salivary glands and cause sequence-specific mRNA degradation as well as a decrease in the histamine-binding capacity of the saliva. This indicated that dsRNA spreads from the site of injection to the various tissues—in this case, the salivary glands. HBP dsRNA–injected ticks showed aberrant feeding behavior, indicating the functional importance of HBP for ticks to be effective feeders. The next report of dsRNA-mediated silencing in ticks was in another ixodid tick, Ixodes scapularis.26 I. scapularis transmits several pathogens, including those that are the causative agents of Lyme disease and human granulocytic ehrlichiosis. Here as in Amblyomma americanum, salivary secretions play a crucial role in the prolonged feeding and successful transmission of pathogens. In the second report, Narasimhan et al. suppressed the expression of the presumed anticoagulant salivary-gland protein (Salp14) and other structural paralogs of Salp14, and reported the functional requirement of this anticoagulant protein in normal tick feeding. The authors designed a dsRNA for Salp14 and were able to silence the entire family of paralogs of Salp 14. This, they suggested, is due to off-target silencing of the siRNA. This is consistent with previous reports, which indicated that RNAi is not absolutely target specific but is siRNA specific.27 One siRNA could silence the target gene and also genes with regions that have greater than 80% similarity to the siRNA sequence. Since the RISC includes an RNA of 21–26 nt, it is not surprising that genes with similarities of this extent could be silenced. Further investigations are required in this aspect of RNAi, and we will not be discussing this aspect in detail as it is beyond the scope of the review. Most recently, tick v-SNARE- and t-SNARE-associated proteins in salivary glands of the lone star tick have been successfully targeted and silenced, and shown to be important for tick feeding.28,29 SNARE and SNARE-associated proteins have been demonstrated to be important in the process of calcium-dependent regulated exocytosis in secretory tissue.30,31 Since salivary glands are the major organs of secretion, and secretion from the salivary glands is calcium sensitive, it was hypothesized that the process of protein secretion involves the participation of these proteins. SNARE proteins were identified to be present within the TSGs of ticks.32 The authors were able to utilize RNAi to test and confirm this hypothesis. An EST project in female A. americanum salivary glands conducted in our laboratory resulted in 16 different cDNAs with significant similarities to Kunitz bovine pancreatic trypsin inhibitors, some with putative anticoagulation activities. To examine their effect on hemostasis, we incubated the tick salivary glands with dsRNA specific for each of these cDNA sequences and monitored the coagulation time against mock controls. Following this approach we could identify two polypeptides with significant effect on hemostasis using sheep plasma (Figure 4A4A).). The dsRNA specific for contig 2 resulted in a significant reduction in the anticoagulant activity, suggesting the putative translated protein of contig 2 to be an anticoagulant. An opposite result was obtained when using the dsRNA specific for contig 9, suggesting this factor to be procoagulant, because the RNAi effect resulted in increasing the anticoagulant activity. The levels of contigs 9 and 2 transcripts, measured by reverse transcription polymerase chain reaction (RT-PCR), were reduced when the salivary glands were incubated with the specific dsRNAs, relative to the controls, where another dsRNA, specific for contig 16, was incubated with the tissues (Figure 4B4B).). In all the above studies, authors have successfully employed RNAi to test and identify the roles of proteins hypothesized to be important for tick feeding.
In a report of the use of RNAi in the hard tick Haemaphysalis longicornis, Miyoshi et al. introduced in vivo a dsRNA for a serine protease and observed a reduction in the transcript levels of the gene encoding for the serine protease.33 Additionally, they were able to observe a 39% reduction in average weight of the ticks injected with the serine protease dsRNA when compared to ticks injected with buffer. It was hypothesized that reduction in size accompanied by prolonged attachment on the host might be due to an inability of the ticks injected with the dsRNA to digest the host blood meal. More recently, de la Fuente et al. applied RNAi in I. scapularis to successfully identify tick protective antigens.34 They injected unfed ticks with groups of dsRNA that were synthesized from cDNA pools identified to encode for tick protective antigens. By performing this, they were able to identify two pools of cDNA that encode tick protective antigens. This study opens up an area of tremendous therapeutic potential of RNAi in tick research. Similar studies could be performed in other tick species to attain valuable information on proteins that are important for the success of tick feeding and to identify tick protective antigens. In the following section, we will be overviewing methodologies involved in performing an RNAi experiment.
The following section briefly outlines the methods involved in a typical RNAi experiment performed in tick salivary glands. For more detailed information, readers are encouraged to refer to the reference list.25,26,28,29
A. americanum lone star female unfed ticks were fed on sheep following the methods of Patrick and Hair.35 Ticks were removed from sheep when they weighed 50–200 mg after about 11–15 days of placement on the host. This weight range, often termed the partially fed state, represents a state of significant growth from its unfed state (4 mg), and a state after which ticks undergo rapid (24 h) growth and engorgement to attain repletion. Tick salivary glands were dissected within 4 h after removal from the host. Tick salivary glands were dissected in ice-cold 100 mM 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer containing 20 mM ethylene glycol bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (pH 6.8) and stored at −20°C in RNAlater (Ambion, Austin, TX) before use.
A cDNA fragment of a gene was cloned into a dual promoter (pCR-2.0) vector or single promoter (pCR-2.1) (TOPO-T/A cloning kit) (Invitrogen, Carlsbad, CA). The cloned fragments were sequenced using an ABI 3700 automated DNA sequencer and M13 reverse primer. The sequences were compared for sequence similarities at the amino acid level using the BLAST-X program.
The cloned cDNA fragments were used as templates for the in vitro synthesis of dsRNA. The plasmid DNAs were linearized with an appropriate restriction enzyme and then used as a template for in vitro transcription using a MEGAscript kit (Ambion), following the manufacturer’s instructions. If using a vector with a single promoter, the plasmids with the gene of interest in opposite orientations were linearized with the same restriction enzyme and used as templates for in vitro transcription. However, it is more convenient to use a vector with two phage promoters. In this case, the plasmid DNA was digested with two different restriction enzymes to linearize the plasmid. The recognition sites of the restriction enzymes used were on the opposite end of the insert to be transcribed from the phage promoter. The linearized plasmids were then used as templates for in vitro transcription using the two promoter-specific RNA polymerases. The two ssRNAs were mixed together and incubated at 65°C for 5 min and later left to anneal at room temperature for 6 h.
Left salivary glands from at least eight partially fed female ticks weighing 50–200 mg were incubated with M199/MOPS buffer. Right salivary glands from the same eight ticks were incubated with at least 8 μg dsRNA. The incubations were carried out for 6 h at 37°C. As a negative control, in a separate experiment, right salivary glands from eight partially fed female ticks weighing 50–200 mg were incubated with a dsRNA unrelated in sequence to the gene of interest. Left salivary glands were incubated as before with buffer.
At least 1 μg dsRNA in 1 μL injection buffer or 1 μL injection buffer alone were injected posteriorly using a 31-gauge needle into 30 unfed female experimental and control ticks, respectively. Injected ticks were kept over a 37°C water bath overnight to check for mortalities or injury due to injection. The ticks were then fed on sheep with the control and experimental ticks on adjacent cells on the same sheep.
After incubation with the dsRNA, total RNA was isolated from both the buffer-incubated and dsRNA-incubated glands using the RNAqueous total RNA isolation kit (Ambion). The RNA was reverse transcribed and the cDNA was used as a template in RT-PCR. The transcript levels of both the gene of interest and an unrelated gene were compared. In the case of the negative control, wherein the glands were incubated with a dsRNA unrelated in sequence to the gene of interest, the transcript levels of the unrelated gene and the gene of interest in the buffer-incubated and dsRNA-incubated glands were compared. It was demonstrated that the dsRNA-mediated silencing is absolutely sequence specific, with the dsRNA designed based on one gene sequence silencing only the gene of absolute sequence similarity. This was confirmed by performing the negative control experiment. In this case, the dsRNA for an unrelated gene silenced only that gene and not the gene of interest.
In another experiment, salivary glands incubated with buffer and dsRNA were homogenized in ice-cold phosphate-buffered saline extraction buffer (1 mM dithiothreitol, 2.5 mM EGTA, 1X Complete Mini protease inhibitor cocktail) (Roche, Indianapolis, IN). Homogenized salivary glands were sonicated for 1 min. Tick salivary gland protein extracts (50 μg each) were loaded onto a 10% SDS-PAGE and subsequently used for a Western blot with antibody to the protein of interest.
Similar experiments were performed in vivo. In a typical in vivo experiment, dsRNA-injected and buffer-injected ticks were fed simultaneously on adjacent cells on the same sheep. Once they attained the partially fed stage of tick feeding (50–200 mg), ticks were forcibly removed from the host; total RNA and protein were extracted from dissected salivary glands and used for similar studies as done in vitro. Other ticks were allowed to feed to repletion and were monitored for aberrant phenotypes (Figure 5A and 5B5B).
In addition to the above methods, one could use quantitative real-time PCR, Northern blotting, and confocal microscopy to confirm the silencing effect of RNAi. Recently, quantitative real-time PCR has largely replaced the traditional RT-PCR due to its increased sensitivity and precision.
The functional importance of a protein within salivary glands could be understood by monitoring for aberrant phenotypes. However, an aberrant phenotype does not give sufficient information to know the actual function performed by the protein of interest. Therefore, it is extremely important to perform functional studies to actually demonstrate the significance of the protein within salivary glands. Unfortunately, this might be relatively difficult to perform for certain proteins in the tick salivary glands due to the lesser information available on tick proteins and genes. Since this procedure varies from protein to protein, we are unable to overview the methods involved in this experiment.
The activated partial thromboplastin time related to the intrinsic pathway was used to examine the anti- or pro-coagulant activities. Twenty microliters of diluted salivary gland sonicated extracts in TC-MOPS or the buffer alone were preincubated with 50 μL of citrated sheep plasma (Sigma; Cat. No. P-4389) for 15 min with continuous shaking in a 96-well microtiter plate. After the preincubation, 100 μL of Alexin (Sigma; Cat. No. A 1801) and 20 mM CaCl2 (1:1) were added and A405nm was measured in a kinetic mode until all samples coagulated. The time from when the reagents were added to when absorbance reached a plateau was counted as the coagulation time.
The protein concentration of the tick salivary gland protein was determined by the method of Bradford with Bio-Rad protein assay dye. Bovine serum albumin was used as the standard protein. Student’s t-test was used to test the significance of the differences between control and experimental groups from three independent incubations with dsRNA, with a P < 0.05 considered as significant.
Total RNA from salivary glands was isolated using RNAqueous (Ambion) and was reverse transcribed according to a standard protocol (Invitrogen). For each group, the cDNA was PCR amplified using gene-specific primers (GSP) for #16 (5′-ttgctgcttcgtattcgttgg-3′ and 5′-cacacaagtagggaaatgtcggc-3′), #09 (5′-gttgggaaccacgctgtcag′ and 5′-ccaggcattttgaatcggg-3′), #02 (5′-tggtgatggtatttgcggcg-3′ and 5′-gcttggttttggaagtaaggtcc-3′), and human β-actin (Stratagene, La Jolla, CA) with a PCR program; 95°C for 2 min, and 28 cycles of 94°C for 60 sec, 55°C for 60 sec, and 72°C for 90 sec, with final 20 min at 72°C.
Ixodid ticks have long been known to be effective feeders that feed for extended periods on the host. But little is known about how ticks overcome the host immune response and manage to stay attached to the host for the entire duration of feeding. However, it has been established that salivary secretions are extremely important for the ticks to be successful feeders and transmitters. Identification of the proteins involved in this process has been slow due to the high sequence variation of proteins in ticks compared with other species. With the advent of high-throughput genomic and proteomic technologies, the availability of sequence information of proteins and genes that encode them has increased significantly. It is extremely important to study the functions performed by these proteins. RNAi, over the past few years, has been an effective and rapid tool in studying the functional roles of the identified proteins in several species. RNAi has been successfully employed and tested in tick salivary gland research. RNAi research in ticks is still in the early stages. Several proteins thought to be important for ticks to be effective feeders and vectors of pathogens could be effectively targeted and silenced. A tick genome sequencing project is currently underway, and once completed will result in a wealth of sequence information of genes expressed in ticks. The significance of the proteins encoded by the expressed genes in ticks has to be studied. RNAi could be highly useful in this regard in tick research. This might provide greater insights into tick biochemistry and physiology, and more importantly into tick control. The proteins determined to be functionally important could be targeted for designing drugs, thereby making ticks less pathogenic.