For discovery of subolesin-interacting proteins, subolesin was used as a bait to search for preys in Rhipicephalus (Boophilus) microplus using the yeast two-hybrid system. Two sequences, GI and GII, were identified that encoded for candidate subolesin-interacting proteins. These sequences were represented in 50% and 10% of the positive clones, respectively. BLAST analysis of the GI sequences did not result in identity to known sequences, except for the EST910636 from a R. microplus cDNA library. However, a transduction/transcription domain was found within the GI open reading frame (ORF) (Fig. 1). The GII sequence was 99% identical (Expect = 4e-102) to Amblyomma sp. elongation factor-1 alpha (EF-1a; [Genbank:AAK12647]) and to other proteins containing EF1_alpha_II and EF1_alpha_III domains (Fig. 2). Both GI and GII sequences contained multiple N-myristoylation and casein kinase II (CK2) and protein kinase C (PKC) phosphorylation sites (Figs. 1 and 2).

The proteins encoded by GI and GII were expressed in Escherichia coli fused to a 6xHis tag with molecular weights of 10 and 22 kDa, respectively and the interaction of these proteins with subolesin was confirmed by co-affinity purification on Ni-NTA columns (Fig. 3). In the absence of either GI or GII recombinant proteins or in the presence of an unrelated His-tagged protein, co-purification of subolesin did not occur, confirming the specificity of the interaction between subolesin and GI or GII (Fig. 3 and data not shown).

RNAi was used to study the effect of the knockdown of subolesin-interacting protein-coding genes, GI and GII, on female tick survival, feeding, oviposition, hatching and egg development. Subolesin, GI or GII dsRNAs or injection buffer (saline control) were injected into either unfed or replete R. microplus female ticks. The injection of GI dsRNA did not affect tick survival, feeding and oviposition, a fact that correlated with the absence of GI knockdown in ticks (Table 1). Although GI expression was reduced in tick eggs, it did not affect hatching (Table 2). Gene knockdown was demonstrated for GII and subolesin in both ticks and eggs (Tables 1 and 2). GII knockdown produced a phenotype similar to that obtained with the silencing of subolesin expression. The injection of GII dsRNA in unfed ticks resulted in reduced tick survival, feeding, oviposition and hatching when compared to control ticks (Table 1). When GII dsRNA was injected into replete ticks, hatching and egg development were affected (Table 2 and Fig. 4). Undifferentiated egg masses were observed in eggs oviposited by replete ticks injected with GII and subolesin dsRNA when compared to saline injected controls (Fig. 4).

The results of the subolesin-protein interactions and RNAi experiments with GI and GII, encoding for subolesin-interacting proteins, suggested that subolesin may be involved in gene expression in ticks. Therefore, the subsequent experiments were directed towards analyzing the effect of subolesin knockdown on the tick gene expression profile.

In the first experiment, the kinetics of subolesin silencing by RNAi was investigated in unfed I. scapularis female ticks. The results showed that over 90% silencing of subolesin transcription was obtained 6 days post injection (dpi) and continued until at least 11 dpi (Fig. 5). Significant differences (P = 0.02) in tick weight between subolesin dsRNA (78 ± 33 mg) and saline injected controls (142 ± 47 mg) were observed in ticks that completed feeding on the host in the presence of males at 11 dpi.

In the second experiment, unfed I. scapularis female ticks were injected with subolesin dsRNA or injection buffer alone. Ticks were then placed on a sheep without males and collected at 6 and 9 dpi, by which time subolesin knockdown was regarded as significant based on the RNAi kinetics experiment (Fig. 5). Subolesin knockdown was corroborated by real-time RT-PCR as 81 ± 3% and 79 ± 5% silencing at 6 and 9 dpi, respectively. A group of ticks collected after 10 days of feeding with males was used to corroborate the effect of subolesin knockdown on tick weight, giving results similar to those obtained in the RNAi kinetics experiment described above. The ticks collected at 6 and 9 dpi were then dissected and used to extract RNA for suppression-subtractive hybridization (SSH) and microarray construction and analysis.

The SSH libraries constructed with the RNA of subolesin dsRNA and saline injected ticks collected at 9 dpi were used for random clone amplification and microarray construction. This microarray was enriched for genes differentially expressed after subolesin knockdown. The microarray was hybridized with total RNA prepared from subolesin dsRNA- and saline-injected ticks at 6 and 9 dpi to evaluate how the expression pattern of candidate differentially expressed genes varied at two different time points after subolesin knockdown (Table 3). The results revealed 34 unique (not redundant) genes that were down or up-regulated by at least two-fold after subolesin knockdown at 6 or 9 dpi (Fig. 6). Of these genes, 28 were down-regulated and 6 were up-regulated after subolesin RNAi. Of the down-regulated genes, 4 were down-reglated at 6 dpi, 20 at 9 dpi and 4 at both 6 and 9 dpi (Fig. 6). Up-regulated genes were found at 9 dpi only (Fig. 6). In the microarray analysis, subolesin expression was silenced by approximately 35% at both 6 and 9 dpi. Although 62% of the unique differentially expressed genes did not have homology to available sequences or were identical to genes with unknown function, the remaining sequences indicated that various biological processes were affected by subolesin knockdown. These included regulation of protein and nucleic acid metabolism (54% of protein ontology assignments), energy pathways (15%), immunity (15%), cell communication and signal transduction (8%) and transport (8%).

Three of the down-regulated genes after subolesin knockdown, identical to I. scapularis putative secreted salivary WC peptide [Genbank:AAY66498] and putative secreted protein [Genbank:AAM93633] and Macrobrachium rosenbergii copper/zinc superoxide dismutase (Cu-Zn SOD) [Genbank:AAZ29240], were selected to corroborate the results of the microarray analysis by real-time RT-PCR. The results demonstrated that the expression of these genes was silenced after subolesin RNAi by 98% and 100% (for [Genbank:AAM93633]), 41% and 59% (for [Genbank:AAY66498]) and 93% and 74% (for [Genbank:AAZ29240]) at 6 and 9 dpi, respectively. Ticks injected with an unrelated 4A8 dsRNA control did not show silencing of these genes after RNAi. The possibility of subolesin RNAi off-target effects was analyzed and complementary 7 bp regions were not found between all possible 20–22 bp subolesin siRNAs and tick cDNA sequences of differentially expressed genes identified in the microarray analysis.

I. scapularis subolesin [Genbank:AAV67031] and human ortholog C6orf166 [Genbank:NP_060534] proteins were compared to predict conserved post-translational modifications. Three conserved PKC phosphorylation sites were found (Fig. 7), which were also present in all known tick subolesin protein sequences (data not shown).

I. scapularis female ticks were obtained from the laboratory colony maintained at the Oklahoma State University tick rearing facility. Off-host ticks were maintained in a 12 hr light: 12 hr dark photoperiod at 22–25°C and 95% relative humidity. R. microplus female ticks (Mozambique strain) were reared in Holstein-Friesian cattle at the Utrecht Centre for Tick-borne Diseases, Utrecht University. Animals were housed with the approval and supervision of the respective Institutional Animal Care and Use Committees.

R. microplus eggs were used for RNA extraction and cDNA library construction. cDNA synthesis and amplification was performed using the Super SMART System™ principle (Clontech Laboratories, Mountain View, CA, USA) with adapted anchor primers containing unique SfiI restriction sites for directional cloning (SMART IV: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATGGAGGCCGGG-3'and CDS III: 5'-ATTCTAGAGGCCTCCATGGCCGACATG(T)30VN-3'). Amplified cDNA was polished using the protocol described by the SMART PCR cDNA synthesis manual (Clontech Laboratories), purified using the DNA Extract II Kit (Macherey-Nagel, Düren, Germany) and subjected to directional cloning in pACT2 plasmid via the SfiI sites. Repetitive electroporation of E. coli JM109 cells was performed to obtain a library with a titer exceeding 3 × 10⁷ cfu/ml.

The yeast two-hybrid screen was performed as dictated in the MATCHMAKER Two-Hybrid user manual (Clontech Laboratories). Full-length subolesin cDNA from R. microplus was inserted in yeast expression vector pAS2_1 at NdeI and PstI sites. Yeast strain AH109 was co-transformed with plasmid pAS2_1_subolesin (bait) and pACT2 R. microplus egg cDNA library (prey) in sequential transformation procedure. The co-transformants were selected after incubation at 30°C for 5 days on synthetic defined (SD) minimal medium lacking tryptophan and leucine. All colonies were pooled from the plates and replated on SD minimal medium lacking tryptophan, leucine, histidine and adenine. Positive clones were analyzed for β-galactosidase activity by colony-filter assays. Prey plasmids of positive clones were rescued using E. coli KC8 and subjected to DNA sequencing.

The GI and GII cDNA fragments, encoding for putative prey proteins interacting with the subolesin bait, were amplified by PCR using oligonucleotide primers QEGI5: 5'-GGCCATGGAGGCCGGGATAGGA and QEGI3: 5'-GGAGATCTATGCTCATTAAGACAAATCTC, and QEGII5: 5'-GGCCATGGCCTCAACCAGGCCCACGGACAAACCCCTC and QEGII3: 5'-GGAGATCTGGCGACCGTTTGCCTCATGTC, respectively and the Access RT-PCR system (Promega, Madison, WI, USA). The PCR primers introduced NcoI and BglII restriction sites for cloning into the expression vector pQE-60 fused to a 6xHis tag (Qiagen Inc., Valencia, CA, USA). The full-length cDNA of I. scapularis subolesin was inserted into the expression plasmid pFLAG-CTC (Sigma, St. Louis, MO, USA) as described previously 8. Recombinant subolesin, GI and GII proteins were expressed in E. coli JM109 as described previously 8. For co-affinity purification of subolesin-interacting proteins, the Ni-NTA spin columns (Qiagen) were used following manufacturer's protocol. The supernatant of GI and GII cell lysates were loaded onto the columns. The columns were washed three times with washing buffer (Qiagen) before loading the purified subolesin. The columns were incubated at 4°C for 4 hours to allow protein-protein interaction and then washed again. The proteins were eluted with elution buffer (Qiagen) and the eluted proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and immunoblotted with anti-subolesin antibodies 8. Controls included affinity purification of GI and GII in the absence of subolesin and loading subolesin onto Ni-NTA columns in the absence of His-tagged GI and GII proteins and in the presence of an unrelated His-tagged protein. GI and GII proteins were detected by Western blot using the HisDetector Western Blot Kit HRP (KPL, Gaithersburg, Maryland, USA).

Oligonucleotide primers homologous to I. scapularis and R. microplus subolesin containing T7 promoters were used for in vitro transcription and synthesis of subolesin dsRNA as described previously 1012, using the Access RT-PCR system (Promega) and the Megascript RNAi kit (Ambion, Austin, TX, USA). For the synthesis of R. microplus GI and GII dsRNAs, oligonucleotide primers GI5: 5'-ATGGAGGCCGGGATAGGA and GI3: 5'-TGCTCATTAAGACAAATCTC, and GII5: 5'-GGCCCACGGACAAACCCCTC and GII3: 5'-CGACCGTTTGCCTCATGTC were used, respectively, with the same procedure described above. The dsRNA was purified and quantified by spectrophotometry.

Unfed I. scapularis female ticks were injected with approximately 0.5 μl dsRNA in the lower right quadrant of the ventral surface of the exoskeleton of ticks 10. Freshly molted R. microplus females were placed on double-sided sticky tape with the ventral side upwards and injected with approximately 0.5 μl dsRNA into the anal aperture using a syringe mounted on a MM3301-M3 micromanipulator (World Precision Instruments (WPI), Berlin, Germany) and connected to an UMPII syringe pump (WPI) 12. Engorged R. microplus females were injected with 5 μl of dsRNA (5 × 10¹⁰–5 × 10¹¹ molecules/μl) in the right spiracular plate within 6 hours after dropping off the host 12. The injections were done using a 10 μl syringe with a 1 inch, 33 gauge needle (Hamilton, Bonaduz, Switzerland). Control ticks were injected with injection buffer (10 mM Tris-HCl, pH 7, 1 mM EDTA) alone (saline negative control). We demonstrated previously that there is no difference between using an unrelated dsRNA or injection buffer alone for negative control in tick RNAi experiments 10.

Two RNAi experiments were conducted with I. scapularis. In the first experiment, designed to determine the time after dsRNA injection in which subolesin knockdown occurs, 20 female ticks per group were injected with subolesin dsRNA or injection buffer. The ticks were held in a humidity chamber for 1 day after which they were allowed to feed on a sheep for 10 days. Three ticks from each group were collected at 1, 3, 6, 9 and 11 days post injection (dpi). Only the ticks collected after 10 days of feeding (11 dpi) were fed together with male ticks to evaluate the effect of subolesin knockdown on tick weight. After collection, ticks were weighed and the tick weight was compared between subolesin dsRNA-injected and saline controls by Student's t-test (P = 0.05). Internal organs were then dissected and stored in RNALater (Ambion) for RNA extraction to determine subolesin mRNA levels by real-time RT-PCR. In the second experiment, 100 I. scapularis female ticks per group were injected with subolesin dsRNA or injection buffer alone. The ticks were held in a humidity chamber for 1 day and then allowed to feed on a sheep without males for 5 and 8 days (6 and 9 dpi) in groups of 50 dsRNA- and 50 saline-injected ticks each. Ten additional female ticks per group were fed with males for 10 days to compare the weight of replete dsRNA-injected and control ticks after RNAi by Student's t-test with unequal variance (P = 0.05). Unattached ticks were removed two days after infestation. After feeding, ticks were removed from the sheep and dissected for RNA extraction.

For RNAi in R. microplus, unfed and replete female ticks were injected with GI dsRNA, GII or subolesin dsRNA or injection buffer alone as described previously 12. In the experiment with unfed ticks, 60–76 freshly molted female ticks were injected and fed on a calf for 15 days with untreated males to determine female tick mortality, weight, oviposition and hatching. Tick mortality was evaluated as the ratio of dead female ticks 15 dpi to the total number of female ticks placed on the calf and was compared between dsRNA and mock-injected control ticks by χ²-test as implemented in Mstat 4.01 (α = 0.01). Ticks completing feeding and egg masses oviposited by each tick were weighed individually and the average ± SD were calculated and compared between dsRNA and saline injected control ticks by Student's t-test with unequal variance (P = 0.05). In the experiment with replete female ticks, 6 ticks were used per group. Each tick was weighed before injection and stored individually in 2 ml Eppendorf tubes with pierced lids in an incubator at 27°C and 95% relative humidity to evaluate oviposition and hatching after RNAi. The weight of replete ticks before injection and the eggs mass per tick were compared with the control saline injected ticks by Student's t-test with unequal variance (P = 0.05). mRNA levels of RNAi targeted genes were determined by real-time RT-PCR. For real-time RT-PCR, internal organs were dissected and total RNA was extracted from 6 females of each dsRNA- or saline-injected group after 6 days of feeding and from 100 mg eggs oviposited by injected replete ticks.

Total RNA was isolated from pooled guts and salivary glands of I. scapularis female ticks injected with subolesin dsRNA or injection buffer at 9 dpi (8 days of feeding) using TriReagent (Sigma) according to manufacturer's instructions. RNA quality was assessed by gel electrophoresis. SSH was performed at Evrogen JCS (Moscow, Russia) as described previously 628. Tester and driver RNAs were subtracted in both directions to construct two SSH libraries enriched for differentially expressed cDNAs in subolesin dsRNA-injected (reverse-subtracted) and saline-injected (forward-subtracted) ticks. Approximately 100 clones from each library were randomly picked up and subjected to differential hybridization with subtracted and non-subtracted probes using the PCR-select differential screening kit (Clontech, Palo Alto, CA, USA), which resulted in > 95% candidate differentially expressed cDNAs.

Tick cDNA fragments (384 from each of the reverse and forward subtracted SSH libraries) were amplified by PCR using pAL-16 vector-specific primers, purified (MultiScreen PCR plates, Millipore, Billerica, MA, USA) and arrayed onto gamma amino propyl silane coated GAPS II slides (Corning, Lowell, MA, USA). Eight pools of 12 clones each from an unsubtracted I. scapularis cDNA library 28 and subolesin cDNA were also arrayed and used to validate normalization. Total RNA was prepared from subolesin dsRNA- and saline-injected ticks at 6 and 9 dpi (5 and 8 days of feeding) using the RNeasy Mini Kit (Qiagen) including the on-column DNA digestion with the RNase-free DNase set following manufacturer's instructions. Total RNA (5 μg) were labeled using the 3DNA Array900 kit with Alexa Fluor dyes (Genisphere, Hatfield, PA, USA), Superscript II (Invitrogen, Carlsbad, CA, USA), the supplied formamide-based hybridization buffer and 24 × 60 mm LifterSlips (Erie Scientific, Portmouth, NH, USA) according to the manufacturer's (Genisphere) instructions. Hybridization signals were measured using a ScanArray Express (PerkinElmer, Boston, MA, USA) and the images were processed using GenePix Pro version 4.0 (Axon, Union City, CA, USA). Ratios were calculated as subolesin dsRNA-injected ticks versus saline-injected control ticks. Normalized ratio values obtained for each probe were averaged across 50 biological replicates and four technical replicates and significant differences were defined as displaying an expression fold change greater than 2-fold. All the microarray data were deposited at the NCBI Gene Expression Omnibus (GEO) under the platform accession number GPL6394 and the series number GSE10222.

The RNA samples prepared as described above from I. scapularis female ticks and from R. microplus female ticks and eggs after RNAi experiments were used for real-time RT-PCR analysis. The RNA from I. scapularis female ticks injected with an unrelated 4A8 dsRNA 29 was used as a control in the analysis of mRNA levels for selected differentially expressed genes after subolesin RNAi. Two primers were synthesized based on the sequences determined for subolesin, GI, GII and three differentially expressed genes after subolesin RNAi (identical to I. scapularis putative secreted salivary WC peptide [Genbank:AAY66498] and putative secreted protein [Genbank:AAM93633] and M. rosenbergii Cu-Zn SOD; [Genbank:AAZ29240]) and used for real-time RT-PCR analysis of mRNA levels in dsRNA- and control ticks. Real-time RT-PCR was done using the QuantiTec SYBR Green RT-PCR kit (Qiagen, Valencia, CA, USA) and a Bio-Rad iQ5 thermal cycler (Hercules, CA, USA) following manufacturer's recommendations and oligonucleotide primers and PCR conditions described in Table 4. mRNA levels were normalized against tick β-actin using the comparative Ct method. mRNA levels were compared between dsRNA-infected and control ticks by Student's t-Test (P = 0.05).

The sequences of positive clones in the yeast two-hybrid screen were sequenced and searched for homology with the PSI-BLAST algorithm through NCBI web site. Proteins with similar domain architectures were characterized with CDART 3031.

The tick cDNAs identified in the microarray analysis as down or up-regulated by more than two-fold after subolesin RNAi were sequenced and analyzed. Multiple sequence alignment were performed to exclude vector sequences and to identify redundant (not unique) sequences using the program AlignX (Vector NTI Suite V 8.0, InforMax, Invitrogen, Carisbad, CA, USA) with an engine based on the Clustal W algorithm. Searches for sequence similarity were performed with the BLASTX program against the non-redundant (nr) peptide sequence database and the database of tick specific sequences at NCBI and VectorBase. Protein ontology was determined using the protein reference database 32. The possibility of subolesin RNAi off-target effects were analyzed by searching for exact complementarity between the seed region (bases 2–8) of all possible 20–22 bp subolesin siRNAs and cDNA sequences of tick differentially regulated genes identified in the microarray analysis, including the sequence of 3' UTRs when known 23.

The pattern search and predictions for tick GI, GII and subolesin post-translational modifications were done by PIR searching against the PROSITE database 33. The search for human ortholog (C6orf166; Genbank accession number NP_060534) protein-protein interactions was done by STRING 3435.

The nucleotide sequences of GI, GII and the ESTs reported in this paper have been deposited in the GenBank database under accession numbers [Genbank:EU436162, Genbank:EU436163 and Genbank:FD482610–FD482874], respectively.