Insect Molecular Biology (2003) 12(3), 299–305
RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum Blackwell Publishing Ltd.
M. N. Aljamali*, A. D. Bior*†, J. R. Sauer‡ and R. C. Essenberg* *Department Biochemistry and Molecular Biology, and ‡Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA Abstract RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick–host interface. Keywords: RNA interference, dsRNA, ticks, Amblyomma, histamine binding protein. Introduction The exponential surge in genomic databases accumulated through recent genome and expressed sequence tag Received 23 October 2002; accepted after revision 7 March 2003. Correspondence: Dr Richard C. Essenberg, Department Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA. Tel.: 405 7446193; fax: 405 7447799; e-mail:
[email protected] †Current address: The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
© 2003 The Royal Entomological Society
(EST) projects has generated a huge pool of coding sequences, but the functions for many of them are still unknown or poorly understood. Gene identity based merely on similarity with other genes with known functions is only the first in a series of steps that ultimately determine the exact role of a gene product in a specific tissue and/ or organism (Sawyer, 2001). RNA interference (RNAi) is becoming an increasingly powerful post-transcriptional gene silencing (PTGS) technique that is providing insight into gene function (Kuwabara & Coulson, 2000; Elbashir et al., 2001; Hannon, 2002) and consequently the reliability of gene identity raised by homology search of the databases. The RNAi process involves an ATP-dependent production of small, ≈ 21–25 nt, interfering RNA molecules (siRNAs) from double stranded RNA (dsRNA) and is believed to be responsible for targeting and destroying specific mRNAs facilitated by the formation of the RNA-induced silencing complex (RISC). The RISC RNA molecules complementary to the mRNAs seem to work as a guide recruiting a ribonuclease that consequently cleaves only specific mRNAs (Bass, 2001; Bernstein et al., 2001; Grishok et al., 2001; Nykänen et al., 2001; Hammond et al., 2000; Plasterk, 2002). Ixodid ticks are obligate ectoparasites that infest mammals and represent a classic example of vector–host interaction (Bowman et al., 1996). Because of the time of feeding on the host (up to 2 weeks) the factors in ixodid female tick saliva that counter immune, inflammatory and haemostatic responses of the host are of intense interest (Bergman et al., 2000; Paesen et al., 2000; Aljamali et al., 2002; Francischetti et al., 2002). Paesen et al. (1999) identified three sex-specific histamine-binding protein isoforms (HBPs) secreted via the saliva of R. appendiculatus ticks with the potential for sequestering histamine at the feeding site, thereby contributing to the success of tick feeding. Furthermore, Sangamnatdej et al. (2002) recently identified a serotonin- and histamine-binding protein (SHBP) from the salivary glands of Dermacentor reticulatus that is expressed in both male and female ticks and is hypothesized to have dual ability of binding both inflammatory mediators as an evolutionary mechanism reflecting host adaptation. In our group, Bior et al. (2002) cloned a differentially expressed 299
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Figure 1. Amino acid blast alignment between the cloned fragment of male Amblyomma americanum (A.a) HBP and the corresponding part of Dermacentor reticulatus (D.r) SHBP. E-value = 9e−28. Identities = 67/137 (48%). Residues involved in histamine binding are doubly underlined, from Paesen et al. (2000).
gene that was up-regulated in the tick salivary glands (TSGs) upon the infestation of A. americanum males, which significantly matched an SHBP from D. reticulatus with most of the residues involved in histamine binding being conserved (Fig. 1). To test the credibility and applicability of RNAi in ticks and the function of the putative HBP in A. americanum, we used the previous clone to construct an HBP sequence-derived dsRNA and conducted both in vitro and in vivo experiments to monitor the RNAi effect at both the gene expression and /or the phenotypic pattern related to tick feeding. Results In vitro incubation of dsRNA with the tick salivary glands In order to show the applicability of RNAi in vitro, right and left salivary glands from eight female ticks were incubated for 8 h with HBP dsRNA derived from a cDNA fragment (Genbank # BI273564) identified in partially fed male A. americanum (Bior et al., 2002) (Fig. 1) or with a dsRNA derived from an A. americanum salivary gland sequence that has a similarity to a conserved kunitz-type peptidase inhibitor domain and no similarity to HBP (negative control with Genbank # AY246557). Cells incubated with dsRNA for HBP showed 35% less binding ability of labelled histamine than the control (Fig. 2a). A comparable reduction in the histamine binding resulted from diluting the labelled histamine with ≈ 30-fold of unlabelled histamine strongly indicating that the decrease in the radioactivity was specific to the bound histamine (data not shown). Using the same incubation conditions, we also found a considerable decrease in the HBP transcript in the HBP dsRNA-treated group compared with the control as detected by the RTPCR using HBP, control and actin gene specific primers (GSPs) (Fig. 2b). In vivo dsRNA microinjection in the whole tick To evaluate the role of HBP in achieving homeostasis at the feeding site, we microinjected the A. americanum unfed female ticks with HBP dsRNA before they were fed on the
Figure 2. In vitro RNAi in tick salivary glands. (a) Tick salivary glands in control and experimental groups represent the left and right salivary glands from eight partially fed female ticks. Cells were incubated in 300 µl TCMOPS buffer for 8 h at 37 °C with 8 µg of the control or HBP dsRNA. TSG homogenates from control and HBP groups were incubated with 2.0 µM 3Hhistamine. Bars represent the average of four experiments and error bars the standard error of the mean significant at the 95% level (paired sample ttest), P = 0.046; an asterisk indicates a significant difference. All incubations with labelled histamine were done on ice for 2 h. (b) RT-PCR of total RNA extracted from control and experimental TSG using HBP, control or actin gene-specific primers indicated on the left panel.
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RNA interference in ticks
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Figure 3. Phenotype, functional and expression differences in dsRNA-injected ticks. (a) Average weights of ticks removed during the feeding cycle in the first experiment. Unfed female ticks were injected with 1 µl injection buffer or 1 µg of HBP dsRNA and put on the host the next day. Numbers on top of the bars indicate the number of ticks removed each day. (b) Northern blot of 10 µg total RNA from tick salivary glands using digoxigenin-labelled probes specific for HBP and actin. Equal intensities of rRNA on a formaldehyde gel indicate equal loading of total RNA. (c) Histamine binding activity in the salivary glands from ticks injected with 1 µg of the control or HBP dsRNA. Bars represent the average of four experiments and error bars the standard error of the mean significant at the 95% level (paired sample t-test), P = 0.019; an asterisk indicates a significant difference. (d) RT-PCR of total RNA extracted from control and experimental TSG using HBP, control or actin gene-specific primers indicated on the left panel.
host. No mortality resulted from the injection alone as both control and experimental ticks survived for 18 h after injection while being held in a humidity chamber prior to placement on the host. In two experiments, the pattern of the control ticks injected with the buffer alone was comparable to un-injected ticks infested simultaneously on the same host (data not shown) whereas the female tick feeding pattern between the control and experimental injected ticks was significantly different (Figs 3a and 4). Although the daily weight averages of ticks removed off the host in both control and experimental ticks were not significantly different, the number of ticks that fed successfully in the first injection experiment was significantly less in the dsRNA-
injected ticks compared with controls (Fig. 3a). Fifty per cent of the dsRNA-injected ticks were still attached on the host on day 20 post-infestation with an average weight of 32 mg while all control ticks had completed feeding by day 16. The levels of the HBP transcript were less in both the ticks removed during the 16 days and those still attached at 20 days relative to the controls as indicated by the Northern blot analysis (Fig. 3b). On the other hand, the levels of the actin transcript were comparable in all groups. In a second experiment, we noted several apparent inflammatory sites in the infestation cell of the dsRNAinjected ticks (Fig. 4). These possible inflammatory reactions at the feeding sites of the ticks were accompanied
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Figure 4. Inflammation-like manifestation in the infestation cell of HBP dsRNA-injected ticks. White arrows indicate inflammatory lesions and blood exudates that appeared only around the feeding sites of the experimental ticks.
with an apparent cellular infiltration and an overall redness in the infestation cell area. To confirm the absence of a microbial infection, biopsies were taken from an inflammatory site and from a tick feeding site of a control. We found no infecting agents in the stained sections of the biopsies although several regions in the experimental sections appeared more necrotic than their control counterparts (data not shown). In this experiment, tick weights and feeding times in both control and experimental groups were comparable. To confirm the specificity of the RNAi mediated by the HBP dsRNA, we conducted two more in vivo experiments in which we injected the control ticks with another dsRNA with no similarity to the HBP sequence, the same as used in vitro experiments. In these experiments, the phenotypic pattern of the un-injected ticks, the injected control and experimental ticks was comparable except for a more apparent inflammatory reaction around the feeding sites of the HBP-injected ticks. However, the histamine binding ability of the salivary glands was significantly less in the HBP group than the controls (Fig. 3c). This was further supported by the RT-PCR data that showed a reduction in the HBP transcript in the experimental group relative to controls and a similar reduction of the PCR-amplified product of the gene from which the control dsRNA was derived in the controls (Fig. 3d). Discussion The success of tick feeding depends on several factors in their saliva, primarily proteins and prostaglandins (Bowman et al., 1997; Aljamali et al., 2002), by which ticks modulate the host response at the tick–host interface. Deleting or reducing one of these factors might cause an aberrant phenotypic response. We report here a method that may permit more precise assessment of the overall significance of secreted gene products at the parasite–host interface of ectoparasitic ticks. In our work, we hypothesized that ticks have the machinery that processes the dsRNA into siRNAs, which target specific mRNAs as this homeostatic
silencing pathway seems to be conserved in several other eukaryotes (Hannon, 2002). The genes of HBPs are transcriptionally active in ticks (Paesen et al., 1999), which makes their transcripts significant targets for RNAi. Both the difference in feeding and the pronounced decrease in the HBP mRNA indicate the ability of dsRNA to spread from the injection site to the salivary glands. This agrees with in vivo RNAi injection experiments in C. elegans that produced mutant phenotypes in the whole worm and even in its progeny (Fire et al., 1998; Grishok et al., 2001). The group of treated ticks remaining attached with small sizes represents a clear example of an aberrant phenotype possibly resulting from distorting the mechanism by which ticks survive the host reactions. This could be a direct effect of the free histamine at the attachment site that is known to hamper the feeding process (Kemp & Bourne, 1980). The reason that this phenotype appeared in only one in vivo experiment is not clear although it might have resulted from a differential metabolism of the dsRNA in the experimental ticks, tick to tick variation or, more probably, variation factor(s) of the host on which ticks were feeding. The same number of male ticks was used in both control and experimental infestation cells, making the possibility of fewer mating events in the experimental group unlikely. Although further histological work might clarify the nature of the inflammatory-like lesions around the feeding sites of the dsRNA-treated ticks, it is possible that both the inflammation-like macro- and micromanifestations might reflect high local histamine concentrations resulting from less efficient histamine binding capability of the tick saliva. The salivary gland homogenates from the HBP dsRNAinjected ticks had less ability to bind free labelled histamine, probably indicating less HBP levels. This argument is supported by a lower transcript level of the HBP in the experimental ticks shown by the Northern blot and the RT-PCR (Fig. 3b,d). The combined phenotypic, functional and molecular in vivo data support the hypothesis that injected dsRNA can be transported from the injection site to the salivary glands
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 299– 305
RNA interference in ticks where it appears to be functionally active in degrading the HBP message. The data also indicate that the RNAi effect is specific in the tick salivary glands for the mRNA of the same sequence as the dsRNA. It could be inferred from the in vitro incubation with the dsRNA and the reduction in both the histamine binding ability and the level of the HBP mRNA that the dsRNA could be readily taken up by the tick salivary glands in vitro (Fig. 2a,b). In fact, TSGs appear to mediate uniquely the uptake of some macromolecules (Wang & Nuttall, 1999) and the dsRNA appears to be no exception. This ability of TSGs might be a major advantage and precludes the need of adding possibly non-physiological conditions such as transfection (Clemens et al., 2000). The uptake of dsRNA is in accordance with several previous RNAi studies including soaking of whole C. elegans in a solution containing dsRNA (Tabara et al., 1998) or the in vitro incubation of readily transfected Drosophila S2 cells with dsRNA (Clemens et al., 2000), all showing the ability of dsRNA to cross cell boundaries and pass through several types of tissues. We limited the time of incubating the tick salivary glands in vitro due to their degenerative nature after tick detachment off the host (Lomas et al., 1998), which might cause an overall non-specific degradation of cellular transcripts with longer incubations. The results of both in vivo and in vitro experiments strongly support a role of HBP dsRNA in decreasing the HBP transcript levels in the salivary glands. However, a complete silencing of the HBP gene could not be seen in the dsRNA-treated groups. Although it has been reported that the potency of RNAi in C. elegans persists over a significant increase in cell mass (Carthew, 2001), the dsRNA might be diluted and /or turned over in vivo through tick feeding with regard to the ≈ 100-fold increase in the size of the salivary gland upon feeding or as other tick tissues proceed through physiological changes. On the other hand, we used high concentrations of dsRNA in the incubation medium assuming less efficient cellular reactions in vitro compared with the in vivo injection (Tuschl et al., 1999; Carthew, 2001). The RNAi mechanism seems to be highly specific and has evolved in nature to target only identical mRNAs (Elbashir et al., 2001). In our study, we used an A. americanum male-derived dsRNA to initiate RNAi in the female tick, which might address the question of RNAi specificity in ticks. However, the A. americanum male HBP sequence matched the SHBP of D. reticulatus that was shown to be expressed in both male and female ticks (Sangamnatdej et al., 2002). Interestingly, the male A. americanum HBP was more similar to the female than the male of R. appendiculatus HBP, 42% vs. 35% identity, respectively. The previous data, in addition to our interference results, may suggest that the A. americanum male HBP is also expressed in the female salivary glands or the two
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sequences share enough identity to make the male-derived dsRNA effective in the females. To our knowledge, this is the first report of the use of RNAi in ticks to assess the function of specific gene products. The ability to knock out mRNA in the salivary glands in vivo and in vitro opens a wide area of research through which tick researchers may be able to test the functions of unknown proteins or determine the components of putative biochemical pathways (Clemens et al., 2000; Hannon, 2002; Muda et al., 2002). This approach could be extended to identify factors that would appear as potential targets for better tick control and reduction in ticks’ ability to serve as vectors for medical and veterinary pathogens. Experimental procedures Experimental animals Unfed female ticks of A. americanum were reared on sheep at the OSU Central Tick Rearing Facility according to Patrick & Hair (1975). Ticks in the in vivo control and experimental groups were subjectively pulled off the host when their weight fell in the range 50–200 mg. This weight range was chosen because it is significantly higher than the weight of the unfed tick (≈ 4 mg) and it approximates the weight of ticks prior to going into rapid engorgement. TSGs were dissected from partially fed females in 0.1 M morpholinopropanesulphonic acid (MOPS), 20 mM EGTA, pH 6.8 dissection buffer, immersed in RNAlater (Ambion) and stored at −20 °C. dsRNA in vitro synthesis All in vitro experiments were done using a dsRNA derived from a sequence with homology to a conserved kunitz-type peptidase inhibitor domain as a negative control. DNA Plasmids were digested separately with SacI or EcoRV (HBP dsDNA in pCR-II, Invitrogen) and SacI or EcoRI (negative control dsDNA in pTriplEX2, Clontech) restriction enzymes. The linear plasmids were precipitated with ethanol over night. The two ssRNAs with opposite polarities were synthesized using the MEGAscript™ kit (Ambion) where T7 and SP6 RNA polymerases were added to make ssRNA from plasmids digested with SacI and EcoRV for the HBP and with EcoRI and SacI for the control, respectively. Samples were treated with DNase to digest the excess DNA and were further phenol extracted and isopropanol precipitated. The two ssRNAs were incubated in 40 µl injection buffer (10 mM tris and 1 mM EDTA, pH 7.4) for 30 min at 65 °C and then left to anneal at room temperature for 4 h. The dsRNA aliquots were stored in −20 °C until used. dsRNA in vitro incubation with TSG The left and right salivary glands from eight partially fed female ticks were incubated for 8 h at 37 °C with 8 µg of the HBP or the control dsRNA in a total volume of 300 µl of the incubation buffer TC-199 (Sigma) containing 20 mM MOPS, pH 7.0, respectively.
Histamine binding assay 3
H-histamine was prepared from 3H-L-histidine (ARC) using histidine decarboxylase (Sigma) according to a standard method
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(Taguchi et al., 1984). The 3H-histamine product was purified by cationic ion exchange column chromatography (Amberlite GC-50, Sigma), eluted with 0.4 M HCl, extracted with 100% butanol after adjusting the pH to ≈ 10 and dried under N2. After the in vitro incubation, TSGs were sonicated in binding buffer (RIPA; 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg / ml PMSF, 10 µg/ml sodium orthovanadate and 30 µg/ ml aprotinin, pH 7.4) and 100 µg of total SG protein was incubated with a final concentration of 2.0 µM 3H-histamine in a total volume of 200 µl for 2 h at 4 °C. After incubation, the total volume was applied to Nanosep 3K Omega columns (Pall). Samples were centrifuged for 25 min at 12 000g at 4 °C and further washed with 200 µl ice-cold PBS pH 7.4 and centrifuged twice. About 50 µl was retrieved from the top of the columns and the radioactivity was measured after adding 5 ml of the ScintiSafe™ Econo2 scintillation liquid (Fisher). Student’s t-test was used to test the significance of the differences between control and experimental groups from four independent incubations with dsRNA, with a P < 0.05 considered as significant.
Microinjection of HBP dsRNA in vivo One microgram of the HBP dsRNA in 1 µl of injection buffer or 1 µl of the buffer alone or the control dsRNA was microinjected into thirty unfed female ticks in each of the experimental and control unfed groups, respectively. The ticks were fed simultaneously in separate but adjacent infestation cells on the same side of the host with untreated males (10 in each infestation cell). Partially fed females were pulled off according to determined tick weight described above.
RT-PCR and Northern blot Total RNA from salivary glands was isolated using RNAqueous (Ambion) and was reverse transcribed using M-MLV reverse transcriptase according to a standard protocol (Gibco BRL). For each group, the cDNA was PCR-amplified using gene specific primers (GSP) for HBP (forward 5′-TGTAATTAATGGCGTTTGTG-3′ and reverse 5′-TGGGCAGATGAAGGAAGACT-3′ ), negative control (forward 5′-TTGCTGCTTCGTATTCGTTGG-3′ and reverse 5′CACACAAGTAGGGAAATGTCGGC-3′) and human β-actin (Stratagene) with a PCR programme; 95 °C for 2 min, and twenty-eight cycles of 94 °C for 60 s, 60 °C for 60 s, 72 °C for 90 s with final 15 min at 72 °C. About 10 µg of total RNA from the salivary glands dissected from the in vivo injected ticks was run on a formaldehyde gel and the RNA was transferred to a nylon membrane and hybridized with both HBP and actin DNA probes labelled with digoxigenin according to a standard protocol (Roche).
Acknowledgements We thank Dr Roger J. Panciera for taking sheep skin biopsies, Mr Jerry Bowman for his help at the Tick Rearing Facility at OSU and Mr Ryan Bobsein for technical assistance. This study was funded by section 14-33 Animal Health Funds through the Oklahoma Agricultural Experiment Station and by grant IBN 9974299 from the National Science Foundation and grant no. 990-2129 from the United States Department of Agriculture (NRI). Approved for publication by the director, Oklahoma Agricultural Experiment Station.
References Aljamali, M.N., Bowman, A.S., Dillwith, J.W., Tucker, J.S., Yates, G.W., Essenberg, R.C. and Sauer, J.R. (2002) Identity and synthesis of prostaglandins in the lone star tick, Amblyomma americanum (L.), as assessed by radio-immunoassay and gas chromatography/ mass spectrometry. Insect Biochem Mol Biol 32: 331–341. Bass, B.L. (2001) The short answer. Nature 411: 428 – 429. Bergman, D.K., Palmer, M.J., Caimano, M.J., Radolf, J.D. and Wikel, S.K. (2000) Isolation and molecular cloning of a secreted immunosuppressant protein from Dermacentor andersoni salivary glands. J Parasitol 86: 516 – 525. Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Role for a bidentateribonuclease in the initiation step of RNA interference. Nature 409: 363 –366. Bior, A.D., Essenberg, R.C. and Sauer, J.R. (2002) Comparison of differentially expressed genes in the salivary glands of male ticks, Amblyomma americanum and Dermacentor andersoni. Insect Biochem Mol Biol 32: 645 – 655. Bowman, A.S., Coons, L.B., Needham, G.R. and Sauer, J.R. (1997) Tick saliva: recent advances and implications for vector competence. Med Vet Entomol 11: 277–285. Bowman, A.S., Dillwith, J.W. and Sauer, J.R. (1996) Tick salivary prostaglandins: presence, origin and significance. Parasitol Today 12: 388 –395. Carthew, R.W. (2001) Gene silencing by double-stranded RNA. Curr Opin Cell Biol 13: 244 –248. Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B.A. and Dixon, J.E. (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA 97: 6499 – 6503. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001) Functional anatomy of siRNAs from mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 20: 6877– 6888. Fire, A., Xu, S., Montgomerym, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806 – 811. Francischetti, I.M.B., Valenzuela, J.G., Andersen, J., Mather, T.N. and Ribeiro, J.M.C. (2002) Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary glands of the tick, Ixodes scapularis: Identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa /tissue factor complex. Blood 99: 3602–3612. Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D., Fire, A., Ruvkun, G. and Mello, C.C. (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106: 23 –34. Hammond, S.M., Bernstein, E., Beach, D. and Hannon, G.J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293 –296. Hannon, G.J. (2002) RNA interference. Nature 418: 244 –251. Kemp, D.H. and Bourne, A. (1980) Boophilus microplus: the effect of histamine on the attachment of cattle-tick larvae-studies in vitro and in vivo. Parasitology 80: 487– 496. Kuwabara, P.E. and Coulson, A. (2000) RNAi-prospects for a general technique for determining gene function. Parasitol Today 16: 347–349.
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RNA interference in ticks Lomas, L.O., Gelman, D. and Kaufman, W.R. (1998) Ecdysteroid regulation of salivary gland degeneration in the ixodid tick, Amblyomma hebraeum: a reconciliation of in vivo and in vitro observations. General Comp Endocrinol 109: 200–211. Muda, M., Worby, C.A., Simonson-Leff, N., Clemens, J.C. and Dixon, J.E. (2002) Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J 366: 73–77. Nykänen, A., Haely, B. and Zamore, P.D. (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107: 309–321. Paesen, G.C., Adams, P.L., Harlos, K., Nuttall, P.A. and Stuart, D.I. (1999) Tick histamine-binding proteins: isolation, cloning, and three-dimentional structure. Mol Cell 3: 661–671. Paesen, G.C., Adams, P.L., Nuttall, P.A. and Stuart, D.L. (2000) Tick histamine-binding proteins: lipocalins with second binding cavity. Biochim Biophys Acta 1482: 92–101. Patrick, C.D. and Hair, J.A. (1975) Laboratory rearing procedures and equipment for multihost ticks. J Med Entomol 12: 389 –390.
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Plasterk, R.H.A. (2002) RNA silencing: the genome’s immune system. Science 296: 1263 –1265. Sangamnatdej, S., Paesen, G.C., Slovak, M. and Nuttall, P.A. (2002) A high affinity serotonin- and histamine-binding lipocalin from tick saliva. Insect Mol Biol 11: 79–86. Sawyer, T.K. (2001) Genes to drugs. Biotechnique 30: 164– 168. Tabara, T., Grishok, A. and Mello, C.C. (1998) RNAi in C. elegans: soaking in the genome sequence. Science 282: 430 – 431. Taguchi, Y., Watanabe, T., Kubota, H., Hayashi, H. and Wada, H. (1984) Purification of histidine decarboxylase from the liver of fetal rats and its immunochemical and immunohistochemical characterization. J Biol Chem 259: 5214 – 5221. Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P. and Sharp, P.A. (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13: 3191–3197. Wang, H. and Nuttall, P.A. (1999) Immunoglobulin-binding proteins in ticks: new target for vaccine development against a bloodfeeding parasite. Cell Mol Life Sci 56: 286 –295.
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