Insect Biochemistry and Molecular Biology 43 (2013) 1133e1141

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Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb

Molecular characterization of the vitellogenin receptor from the tick, Amblyomma hebraeum (Acari: Ixodidae) Alexander D. Smith*, W.Reuben Kaufman 1 Department of Biological Sciences, University of Alberta, CW 405, Biological Sciences Building, Edmonton, Alberta T6G 2E9, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 July 2013 Received in revised form 1 October 2013 Accepted 4 October 2013

We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Amblyomma hebraeum Low-density lipoprotein receptor Reproduction Ticks Vitellogenin Vitellogenin receptor

1. Introduction In insects, the major yolk protein is vitellin (Vn), a processed form of vitellogenin (Vg), which is synthesized primarily in the fat body. After release into the haemolymph, Vg is taken up into developing oocytes by the vitellogenin receptors (VgRs) located within clathrin-coated pits on the oocyte surface (Sappington and Raikhel, 1998). VgRs are large membrane bound proteins that make up a specialized subfamily of the low-density lipoproteins receptor (LDLR) superfamily (Schneider, 1996; Rodenburg et al., 2006). The molecular characteristics of VgRs have been well described for insects [e.g.: the fire ant Solenopsis invicta (Chen et al., 2004) and the cockroaches Periplaneta americana and Leucophaea maderae (Tufail and Takeda, 2005, 2007)], but have been best characterized, both biochemically and molecularly, in the mosquito Aedes aegypti (Sappington et al., 1995). VgRs have also been described from a number of other arthropods, including crustaceans such as the red mud crab Scylla serrata (Warrier and Subramoniam, 2002), and the shrimp Penaeus monodon (Tiu et al., 2008); as well as chelicerates such as the ticks Dermacentor

* Corresponding author. Tel.: þ1 780 680 9979. E-mail addresses: [email protected], [email protected] (A.D. Smith). 1 Present address: 215 Cormorant Crescent, Saltspring Island, BC V8K 1G8, Canada. 0965-1748/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibmb.2013.10.002

variabilis (Mitchell et al., 2007) and Haemaphysalis longicornis (Boldbaatar et al., 2008). VgRs have also been characterized from vertebrates including chickens (Bujo et al., 1994), the African clawed-toad Xenopus laevis (Okabayashi et al., 1996), and the rainbow trout, Oncorhynchus mykiss (Prat et al., 1998); as well as from a member of the Nematoda: Caenorhabditis elegans (Grant and Hirsh, 1999). As members of the LDLR superfamily, both insect and tick VgRs are characterized by a highly conserved arrangement of various motifs. These modular elements consist of (i) extracellular, ligandbinding LDLR Class A cysteine-rich repeats, (ii) epidermal growth factor (EGF)-like LDLR Class B cysteine-rich repeats, (iii) repeats characterized by a YWXD motif that are proposed to form a bpropeller domain (Springer, 1998)), (iv) a serine/threonine-rich tract, possibly indicative of the O-linked carbohydrate domain present in some LDLR receptor types, (v) a transmembrane domain to anchor the receptor to the cell surface, and (vi) a cytoplasmic tail containing an internalization signal (Sappington and Raikhel, 1998; Tufail and Takeda, 2009). Despite the many studies, reviews and chapters on vitellogenesis in ticks, there is still relatively little data on the molecular processes of vitellogenesis in ticks, as compared to insects. There is even less data on the molecular characteristics of tick VgRs, with sequence data available from only two species e D. variabilis (Mitchell et al., 2007) and H. longicornis (Boldbaatar et al., 2008). Previous studies have shown that yolk uptake is regulated

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differently in D. variabilis and Amblyomma hebraeum (Friesen and Kaufman, 2004; Seixas et al., 2008; Thompson et al., 2005), and that an additional vitellogenin uptake factor (VUF) may be required for yolk uptake in A. hebraeum. To gain more insight into this process, we have determined the full-length cDNA and deduced amino acid sequence of the VgR from the ixodid tick A. hebraeum, described its molecular characteristics and compared its structure to those of other ticks and insects. We also describe the tissue- and temporal-specificity of expression of these genes examined by RTPCR and in situ hybridization, along with the effects of disruption of expression using RNA interference. 2. Materials and methods 2.1. Tick rearing and feeding Ticks were maintained in an incubator (under constant darkness, 26  C, and >95% RH). For feeding, ticks were confined to a cloth-covered foam arena attached with latex adhesive (Roberts 8502 Latex, Bramalea, Ontario, Canada) to the shaven back of a rabbit as described by Kaufman and Phillips (1973). The use of rabbits for all research presented in this study was reviewed and approved by the Biosciences Animal Policy and Welfare Committee, University of Alberta. 2.2. Tissue collection for RNA extractions and in situ hybridization

2.5. Degenerate PCR To obtain the A. hebraeum VgR, degenerate primers were designed by aligning highly conserved regions of VgR protein sequences from D. variabilis (GenBank accession number DQ103506), H. longicornis (AB299015) and Ixodes scapularis (XM_002400052) using BlockMaker (Henikoff et al., 1995), and degenerate primers designed from conserved regions using CODEHOP (Rose et al., 1998). The resulting primers were aligned with D. variabilis sequences, and only those with less than 64-fold degeneracy and produced amplicons of appropriate size for cloning were used (Table 1). Degenerate PCR reactions were performed using 0.5 ml of cDNA template, degenerate primers, and Maxima Hot Start Taq DNA polymerase (Thermo Fisher Scientific). Amplification was carried out using touchdown PCR with the following conditions: 94  C for 4 min, 10 cycles of 94  C for 30 s, 60  C for 30 s, and 72  C for 3.5 min; 10 cycles with an annealing temperature of 59  C; followed by 15 cycles using a 58  C annealing temperature; a final extension was performed at 72  C for 10 min.

Table 1 Primers used for identification and analysis of the Amblyomma hebraeum VgR (AhVgR). Purpose

Engorged female ticks were collected from the host, counting the day of drop-off as day 0 post-engorgement. Females were maintained in the colony incubator until dissection at day 0, 4, 8 or 10 post-engorgement. To collect tissues, the ticks were immobilized on small sterile plastic Petri dishes with a drop of cyanoacrylate glue and chilled at 4  C for a minimum of 30 min to sedate the tick and reduce the likelihood of puncturing the delicate gut while dissecting away the cuticle. Ticks were flooded with 1 phosphatebuffered saline (PBS), dorsal cuticle removed using a micro scalpel, and various organs, including the ovary, midgut, fat body, salivary glands, Malpighian tubules and trachea harvested. Tissues were placed immediately into RNAlater (Ambion, Austin, TX, USA) and stored at 20  C until used for RNA extractions or in situ hybridization.

Sequence (50 e30 )

Degenerate PCR VgR Degen Fwd 1 VgR Degen Rev 1

GCACCAGAACCTGTACTGGGTNGAYGC CGACGTGGTAGTCCATGCCRTRCATRTC

RACE

Abridged primer

GGCCACGCGTCGACTAGTACTTTTTTTT TTTTTTTTT GGCCACGCGTCGACTAGTAC

Gene Specific

VgR Fwd 1 VgR Fwd 2 VgR Fwd 3 VgR Fwd 4 VgR Fwd 5 VgR Fwd 6 VgR Fwd 7 VgR Fwd 8 VgR Fwd 9 VgR Fwd 10 VgR Rev 1 VgR Rev 2 VgR Rev 3 VgR Rev 4 VgR Rev 5 VgR Rev 6 Ah16S Fwd Ah16S Rev

GGATCGCGCCTGCTTCTGC GTTTCTCGATGCACGGCCACG GCTGGCTGAAGACACTCTTGCG CTGCGTAGAATCCGATGACCG GTAGCCTTGCTTGTCCTCGG GTTCCTGCTCTACATGCTTCCG GCTGCGGCGATGGTCAGTG GTCACAACAATGAGTGCATCCC GACTTGGCCGCCGGACTT CATCGGTTCCGTCTTCGGCATG CAGGCGATGCACTCGTGAGATC CCATTGGCTCGGCACACCTC CCACCATTGAGGCAAACCGG GCCGTGCATATCCGCAAGAG CCAGTAGAGGGGAAGGCAC GGTGGCAAGGTCCAAGTACTC CTGCTCAATGATTTTTTAAATTGCTGTGG CCGGTCTGAACTCAGATCAAGT

Sequencing

T7 Fwd SP6 Rev M13 Fwd M13 Rev pJET1.2 Fwd pJET1.2 Rev

TAATACGACTCACTATAGGG TATTTAGGTGACACTATAG GTAAAACGACGGCCAG CAGGAAACAGCTATGAC CGACTCACTATAGGGAGAGCGGC AAGAACATCGATTTTCCATGGCAG

RNAi

T7-VgR Fwd

TAATACGACTCACTATAGGCGGGAGTT CTTTTCGTTCTGAG TAATACGACTCACTATAGGCCAGTAG AGGGGAAGGCAC TAATACGACTCACTATAGGGCTTAATC AGTGAGGCACCTATC TAATACGACTCACTATAGGCATTTCCG TGTCGCCCTTATTC

2.3. RNA isolation RNA was extracted from various tissues or whole ticks using either the RNeasy Mini RNA extraction kit (Qiagen, Valencia, CA, USA) or Trizol (Invitrogen, Grand Island, NY, USA) according to the manufacturers’ directions and contaminating DNA removed using a DNA-Free kit (Ambion), according to the manufacturer’s specifications. RNA quantity and purity was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and then run out on a 0.8% agarose gel for visual confirmation of quality.

Description

Universal primer

2.4. cDNA synthesis

T7-VgR Rev

One mg of total RNA was reverse-transcribed using either random hexamer primers (for degenerate PCR or gene expression analysis), or gene-specific primers (for 50 /30 RACE; see section 2.5) and SuperScript III reverse transcriptase (Invitrogen) to synthesize first-strand cDNA, according to the manufacturer’s recommendations. All gene-specific primers were examined using OligoCalc (Kibbe, 2007) to check for possible self-complementarity, and to ensure that primer pairs had similar melting temperatures.

T7-Bla Fwd T7-Bla Rev In situ probes

VgR antisense probe TAATACGACTCACTATAGGGAGACT TCGGCTGGGTGGACCTC VgR sense probe ATTTAGGTGACACTATAGAAGAGGC TACTGCAACCCTGGTTTC

RACE, rapid amplification of cDNA ends; RNAi, RNA interference.

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2.6. 50 /30 RACE To obtain the 50 -regions of AhVgR, 50 RACE was performed according to protocols in Sambrook and Russell (2001). The 50 -end of VgR was amplified using universal adaptor primers, gene-specific VgR primers (Table 1) and Maxima Hot Start Taq DNA polymerase. Reactions were amplified using a touchdown PCR protocol (1 cycle of 94  C for 5 min; 5 cycles each of 94  C for 40 s, [58  Ce55  C] for 30 s, and 72  C for 4.5 min; 10 cycles of 94  C for 40 s, 54  C for 30 s, and 72  C for 4.5 min). Amplification of the 30 cDNA ends was performed using a 30 RACE System for Rapid Amplification of cDNA Ends kit (Invitrogen) according to the manufacturer’s directions. The 30 -end of AhVgR was amplified as above, with cycling conditions consisting of an initial denaturation at 94  C for 5 min followed by 35 cycles of 94  C for 30 s, 60  C for 30 s and 72  C for 2 min, followed by a final extension at 72  C for 10 min. 2.7. Cloning and sequencing of the putative AhVgR Amplified cDNA fragments were sequenced directly, or after subcloning into a pGEM-T (Promega, Madison, WI, USA), or a pJET1.2 (Thermo Fisher Scientific) vector. The constructed plasmids were transformed into Escherichia coli OmniMAX 2 T1R cells (Invitrogen) and plasmids purified using a High Pure Plasmid Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocols. Sequencing reactions used BigDyeÒ Terminator v3.1 Cycle Sequencing mix (Applied Biosystems, Foster City, CA, USA) and T7, SP6, pJET1.2 forward and reverse sequencing primers and/or gene-specific primers (Table 1).

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microscope. The obtained images were all processed using Adobe Photoshop to adjust colour, brightness and contrast for clarity. 2.10. Sequence and bioinformatic analysis Following the removal of all primer and vector sequences from the raw sequence data, sequences were assembled using GeneTool (Biotools, Inc. Edmonton, Canada), then compared against the nonredundant GenBank nucleotide database using BLASTN (www.ncbi. nlm.nih.gov) searches (Altschul et al., 1997) to determine putative gene identity. Open reading frames (ORFs) were predicted using the NCBI ORF finder, and putative protein sequences compared against the non-redundant GenBank protein database using BLASTP searches (Altschul et al., 1997) to confirm putative protein identity. The NCBI Conserved Domain Database (CDD; Marchler-Bauer et al., 2011) was used to identify conserved domains. Tools available at the Center for Biological Sequence Analysis at the Technical University of Denmark (CBS; http://www.cbs.dtu.dk/) were used to identify signal peptide cleavage sites (SignalIP; Petersen et al., 2011), potential transmembrane regions (TMHMM), as well as possible phosphorylation (NetPhos 2.0) and N-linked glycosylation (NetNGlyc) sites. The GPP Prediction Server (http://comp.chem. nottingham.ac.uk/glyco/) was used to evaluate potential O-linked glycosylation sites (Hamby and Hirst, 2008), and PATTINPROT (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page¼/NPSA/ npsa_server.html) was used to identify specific conserved motifs in the protein sequences (Combet et al., 2000). Tools available at the ExPASy: SIB Bioinformatics Resource Portal (http://web.expasy.org/ ) were used to determine putative molecular weights and isoelectric points (Compute pI/Mw tool), potential sulfation sites (Sulfinator), and domain structure (ScanProsite tool).

2.8. Determination of AhVgR expression by RT-PCR

2.11. Sequence alignments and phylogenetic analysis

Levels of AhVgR mRNA were evaluated by semi-quantitative RTPCR. Total RNA was isolated and cDNA prepared as described in sections 2.3 and 2.4. The cDNAs were diluted 1/10 with sterile, DEPC-treated water and AhVgR sequence amplified using Maxima Hot Start Taq DNA polymerase and VgR Fwd 3 and Rev 3 genespecific primers (Table 1). Cycling conditions consisted of an initial denaturation at 94  C for 5 min followed by 28 cycles of 94  C for 15 s, 55  C for 15 s and 72  C for 1 min followed by a final 10 min 72  C extension.

The AhVgR protein sequence was compared to those of other arthropod VgRs. Sequences were aligned using MUSCLE (Edgar, 2004), and phylogenetic relationships inferred using MEGA5 (Tamura et al., 2011). Distance matrices were constructed according to the JoneseTayloreThornton model (Jones et al., 1992), and trees constructed using the neighbour-joining method (Saitou and Nei, 1987), with bootstrap values assessed at 1000 replicates. Sequences were compared in a pairwise fashion, with all ambiguous positions removed for each sequence pair.

2.9. Determination of AhVgR expression by in situ hybridization

2.12. RNA interference (RNAi)

In situ hybridization protocols were modified from those of Lehmann and Tautz (1994), Niwa et al. (2004), Osborne and Dearden (2005), and Horigane et al. (2010). RNA probes were generated from PCR products with T7 and SP6 ends produced from amplification of ovary cDNA using gene-specific primers (Table 1). Sense and antisense probes were produced using SP6 and T7 RNA polymerase (NEB) respectively and DIG RNA labelling mix (Roche) according to the manufacturer’s specifications. Tissues used for in situ hybridizations were isolated from females 10-days postengorgement, fixed in 4% paraformaldehyde then dehydrated in a graduated series of methanol washes and stored at 20  C. Tissues were re-hydrated in a series of methanol/PBS washes, treated with 5 mg/ml proteinase K (Ambion) and post-fixed in 4% paraformaldehyde. Hybridizations were performed at 65  C overnight in buffer containing 50% formamide. Anti-Digoxigenin-AP Fab fragments (Roche), preabsorbed with powdered, fixed female tissue, and NBT/BCIP (Roche) were used to detect hybridized probe. Following staining, the dissected midgut, trachea, fat body, muscle, cuticle and reproductive tissues were observed with a stereo

AhVgR knock-down was performed using dsRNAs with homology to AhVgR, or b-lactamase (Bla), as a negative control. T7 flanked gene regions were PCR amplified using gene specific primers (Table 1), then purified using the QIAquick PCR purification kit (Qiagen). Purified PCR products were used as template to synthesize VgR- and Bla-dsRNA in vitro using the MEGAscript RNAi kit (Ambion) according to the manufacturer’s specifications. The targeted regions were specifically chosen because A) a BLAST search revealed no significant homology to other genes in GenBank, decreasing the likelihood of off-target effects, and B) analysis of each gene using the IDT RNAi oligo design tool (http://www.idtdna. com/Scitools/Applications/RNAi/RNAi.aspx) indicated the presence of a potential dicer recognition site in the targeted regions. Unfed adult female ticks were divided into weight-matched groups and injected with 1 mg of AhVgR-dsRNA, Bla-dsRNA, or TE buffer control in a total volume of 2 ml. All ticks were marked by tying coloured threads to one or more legs and anchoring it in place with a small drop of cyanoacrylate glue (Loctite Corp.). Injections were made through the fourth coxa, into the haemocoel, using 33G,

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SP LBD1

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AhVgR NH2

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PmoVgR 31% NH2

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AaeVgR 31% NH2

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HlVgR 71% NH2

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DvVgR 78% NH2

EGF precursor 1

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Fig. 1. Schematic comparison of Amblyomma hebraeum VgR (AhVgR) with those of Dermacentor variabilis (DvVgR), Haemaphysalis longicornis (HlVgR), Aedes aegypti (AaeVgR), and Penaeus monodon (PmoVgR). Ligand-binding LDLRA repeats of the lipid binding domains are indicated with numbers 1e8, while EGF-like repeats are marked with roman numerals I and II indicating non-calcium binding and calcium-binding repeats respectively. The YWXD containing repeats that form b-propeller domains are labelled aef. The percentages on the left indicate overall identity of each protein compared to AhVgR. SP, signal peptide; LBD, lipid binding domain; O, potential O-linked sugar domain; TM, transmembrane domain; C, cytoplasmic domain. Sequences were obtained from GenBank: DvVgR (AAZ31260), HlVgR (BAG14342), AaeVgR (AAC28497), and PmoVgR (ABW79798).

½ inch hypodermic needles and 1.00 cc DB Yale Tuberculin syringes (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) placed in a microapplicator (Instrumentation Specialities Co., Lincoln, Neb.; model M). Following injection, ticks were stored overnight in the colony incubator at 26  C, to determine mortality. Equal numbers of ticks from the experimental and control groups, along with an equal number of males, were placed on rabbit hosts. Ticks were monitored daily for mortality, and engorged ticks removed and weighed. Feeding success was determined by measuring numbers of engorged ticks, engorged weights, extent of oviposition and egg hatching success. At 8 days post-engorgement, females were dissected, and tissues harvested. Each ovary was weighed, its developmental stage assessed (Seixas et al., 2008), and the eight largest oocyte lengths recorded. Total RNA was extracted from the ovary of each female and cDNA synthesized to examine dsRNA mediated knock-down. 2.13. Nucleotide sequence accession number Nucleotide sequence data for AhVgR has been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database, accession number JX846592. 3. Results 3.1. Sequence and structural analysis of the AhVgR The full-length AhVgR cDNA is 5703 base pairs (bp), comprising a 99 bp 50 untranslated region (UTR), a 5406 bp open reading frame (ORF) encoding 1801 amino acid (aa) residues, and a 153 bp 30 UTR with a 45 bp poly-A tail occurring 20 bp downstream of the AATAAA polyadenylation signal. The full-length nucleotide sequence has 75% and 73% identity to the D. variabilis and H. longicornis VgRs respectively (DQ103506 and AB299015 respectively; E-values: 0.0). AhVgR also exhibits 67% identity to the partial I. scapularis VgR sequence available in GenBank (XM_002400052). The predicted amino acid sequence of the translated AhVgR ORF is 1801 aa and has a 22 aa predicted signal peptide. Following cleavage of the signal peptide, the mature protein was predicted to

have a molecular weight of 196.5 kDa, with a pI of 5.21. Analysis of conserved domains indicated that AhVgR is a member of the LDLR superfamily, containing the conserved arrangement of five modular elements that characterize this superfamily (Fig. 1). AhVgR possesses twelve LDLR class A (LDLRA) repeats, arranged in two binding domains with four repeats in the first site and eight in the second. Each repeat contains six cysteine residues and a conserved SDE acidic residue region between the fifth and sixth cysteine residues. A total of eight epidermal growth factor (EGF)like domains (class B repeats), each also containing six cysteine residues, are present, two of which are calcium-binding EGF-like domains. Each LDLRA domain is followed by one EGF-like domain and one calcium-binding EGF-like domain. A total of 18 repeats containing a YWXD motif are present in three groups of six. The first two groups are followed by a single EGF-like domain, with the last group followed by two EGF-like domains. A hydrophobic transmembrane region encompassing residues 1670e1692 is followed by a cytoplasmic domain containing potential internalization signals at residues 1742e1747 (following the consensus sequence FXNPXF), and at 1719e1720 and 1754e1755 (dileucine (LL) motif). A putative O-linked sugar domain (OLSD) is predicted in a serine-rich stretch of 17 aa (residues 1653e1669, 29% Ser) between the final cysteine of the EGF-like domain and the beginning of the hydrophobic transmembrane domain. In addition to this potential post-translational modification, 11 putative Nlinked glycosylation sites are recognized with the consensus sequence NX[S/T], and 99 potential phosphorylation sites (serine: 57, threonine: 18, and tyrosine: 24) are predicted in the AhVgR amino acid sequence. Although AhVgR amino acid sequence has relatively high homology to other tick VgRs, the highest homology being to the D. variabilis VgR with 78% identity and 88% similarity, sequence homology was much lower when comparing AhVgR to those from insects. AhVgR has only 32% identity and 48% similarity to the cockroach P. americana VgR. Phylogenetic analysis of amino acid sequences, comparing AhVgR with VgRs from hard ticks, insects and crustaceans indicate that AhVgR groups with other tick VgRs, which themselves form a distinct clade, separate from VgRs present in insects and crustaceans (Fig. 2).

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3.2. Expression of the AhVgR transcript AhVgR message was expressed in adult females that had fed to repletion, and not in males, or unfed or partially fed females (Fig. 3a). Low levels of AhVgR mRNA, compared to a 16S rRNA control, were observed on the day of engorgement, but increased over time, with increased amplification observed in samples collected 4 and 10 days following engorgement. The positive control, 16S rRNA was detected in all samples. Tissue-specific expression of each mRNA was examined by RTPCR of total RNA collected from various tissues from females 0 (d0pE), 4 (d4pE) or 10 days post-engorgement (d10pE; Fig. 3b). AhVgR was expressed in all ovary samples, but was not detected in samples of fat body, midgut or a combined sample of miscellaneous tissues including Malpighian tubules, tracheae, salivary glands, Géné’s organ, synganglion, muscles and cuticular epithelia collected from any of the time points (Fig. 3b). Localization of AhVgR transcripts was further investigated by in situ hybridization of various tissues harvested from engorged females 10 days following engorgement (Fig. 4). The only tissue to exhibit a high degree of staining was the ovary. Ovaries incubated with the antisense probe, but not with the sense probe, showed visible blue staining of the oocytes (Fig. 4aeb). This was most visible in the smallest oocytes, which had not yet begun to fill with yolk particles. Some staining was visible in the larger oocytes, but was much more difficult to discern. Specific staining was not observed using either antisense or sense probes, in fat body, midgut, Malpighian tubules, Géné’s organ, salivary glands, or muscle and cuticle samples (Fig. 4cen). A small amount of nonspecific staining was observed on the cuticle and in the fat body, but no widespread, or localized staining indicative of transcript being located in these tissues. A high level of non-specific staining was seen in the Malpighian tubules. However, this staining was confined to the guanine waste products within the lumen; no staining of Malpighian tubule cells was observed. As this staining of the guanine waste was observed in both the antisense and sense probes, the colour was likely due to a reaction between the

Fig. 3. Spatial and temporal expression of AhVgR as shown by semi-quantitative RTPCR. (a) Total RNA isolated from whole unfed or fed males, and unfed, partially fed, or fully engorged females 0, 4 or 10 days post-engorgement. AhVgR transcript was amplified from only those samples isolated from engorged females. (b) Total RNA isolated from various tissues: other (a pooled mixture of synganglion, muscle, Géné’s organ, Malpighian tubule, trachea, and salivary gland tissues), ovary, fat body or midgut belonging to engorged females 0, 4 or 10 days following engorgement. Ah16S transcript levels were used as an internal control. Abbreviations: p. fed ¼ partially fed to w10 unfed weight; eng ¼ engorgement.

colourimetric detection reagents and the guanine wastes as opposed to a specific staining reaction. 3.3. Gene knock-down of AhVgR by RNA interference Ovaries harvested from females injected with VgR-dsRNA 8 days after engorgement showed decreased yolk uptake (Fig. 5) and had significantly smaller oocytes (359  10 mm) compared to the ovaries of the Bla-dsRNA (545  7 mm) or TE-injected (578  7 mm) controls (p < 0.000). Ovaries from VgR-dsRNA injected females were also

Crustaceans D. melanogaster A. aegypti P. monodon N. lugens

M. rosenbergii

P. americana B. germanica

1000 1000 987

1000 546

H. longicornis

1000

787

I. scapularis

1000

L. maderae

1000

946 1000

1000

950

S. invicta

1000

A. hebraeum D. variabilis

S. litura

1000

Ticks B. mori

A. mellifera

Insects

A. pernyi

Fig. 2. Phylogenetic tree of VgR from various arthropod species. The tree was constructed using the neighbour-joining method, from distance matrices built from amino acid sequences according to the JoneseTayloreThornton matrix-based model, using MEGA5. Branches corresponding to partitions reproduced in fewer than 50% of the bootstrap replicates are collapsed. The units of distance are the number of amino acid substitutions per site. Bootstrap support values from 1000 replicates are indicated at the nodes. Sequences were compared pairwise with all ambiguous positions removed for each pair, resulting in a total of 2369 positions in the final dataset. All non A. hebraeum amino acid sequences were obtained from GenBank. Tick amino acid sequences were obtained from Amblyomma hebraeum (JX846592), Dermacentor variabilis (AAZ31260), Haemaphysalis longicornis (BAG14342), and Ixodes scapularis (XP_002400096). Insect amino acid sequences were from Aedes aegypti (AAC28497), Antheraea pernyi (AEJ88360), Apis mellifera (XP_001121707), Blattella germanica (CAJ19121), Bombyx mori (ADK94452), Drosophila melanogaster (AAB60217), Leucophaea maderae (BAE93218), Nilaparvata lugens (ADE34166), Periplaneta americana (BAC02725), Solenopsis invicta (AAP92450), and Spodoptera litura (ADK94033). Crustacean amino acid sequences were from Macrobrachium rosenbergii (ADK55596), and Penaeus monodon (ABW79798).

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Fig. 4. Localization of AhVgR transcript in mated females 10 days following engorgement as shown by whole tissue in situ hybridization. Dark purple staining indicates AhVgR mRNA expression. Following dissection, tissues were stained with either an antisense probe (a, c, e, g, i, k, m) or a sense probe control (b, d, f, g, j, l, n). Oocytes present in ovaries of mated females stained with the antisense probe were stained dark purple (a) indicating AhVgR expression. Ovaries of mated females stained with the sense probe as a control (b) did not exhibit any staining. No staining was observed in fat body stained with either antisense (c) or sense probes (d), nor was staining observed in the midgut (e, f). The orange substance present in midgut samples is residual gut contents from the tick’s previous blood meal. The majority of the gut contents was removed prior to fixation, but not all could be removed. A high degree of non-specific staining was observed in the waste products present in the Malpighian tubules in both antisense (g) and sense (h) probed samples; however, the cells themselves were not stained in either instance. Géné’s organ (i, j), the salivary glands (k, l) and muscle and cuticle samples (m, n) also did not exhibit any staining with either antisense or sense probes. The yellow colour present in the muscle sample is due to the natural colour of the cuticle. Scale bars ¼ 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

significantly developmentally delayed (p < 0.000). According to the ovarian growth phase (OGP) system developed by Seixas et al. (2008), VgR-dsRNA injected females generally possessed ovaries between OGP stages 2 and 3, whereas the controls generally exhibited ovaries in approximately OGP 4, with some individuals in OGP 5. The ultimate sizes of ovaries and eggs masses produced by VgR-dsRNA injected females were not significantly different from those of the controls, although these females did take

significantly longer to begin oviposition (15  2 days; p ¼ 0.010) compared to the Bla-dsRNA (10  1 days) or TE-injected (10.5  1 days) controls. There were no significant differences in engorged weight or mortality rate between treatment groups. RT-PCR analysis of total RNA extracted from ovaries of VgR-dsRNA females showed a decreased abundance of VgR mRNA compared to Bla-dsRNA and TE-injected controls (Fig. 5). Approximately similar amounts of the 16S rRNA positive control was detected in all samples.

A.D. Smith, W.Reuben Kaufman / Insect Biochemistry and Molecular Biology 43 (2013) 1133e1141

Fig. 5. The effect of disrupting AhVgR mRNA expression via RNA interference on ovaries of mated A. hebraeum females. Ovaries of females injected with AhVgR-dsRNA (a), the non-specific RNAi negative control b-lactamase (b), or the injection buffer (TE) control (c) were dissected from females eight days following engorgement. RNA was extracted from each sample and subjected to semi-quantitative RT-PCR to determine approximate levels of knock-down (d). Ovaries from females injected with AhVgRdsRNA (a) exhibited a small degree of yolk uptake, but all oocytes were still very small and poorly developed, and appeared to be between ovarian growth phase (OGP) 2 and 3 according to the system developed by Seixas et al. (2008). Ovaries from both control groups (b, c) appear to be in OGP4, with the vast majority of oocytes being very well developed, and filled with large yolk granules. RT-PCR analysis indicated that levels of AhVgR mRNA were much lower in AhVgR-dsRNA treated samples than in either control. Levels of Ah16S mRNA were examined as an internal control. Scale bars ¼ 1 mm.

4. Discussion 4.1. VgR structure Similar to both DvVgR and HlVgR, AhVgR has four cysteine-rich LDLRA repeats in the first ligand-binding site, and eight in the second. This is different from most insect VgRs, which generally have a total of 13 LDLRA repeats in a five- and eight-repeat arrangement (Tufail and Takeda, 2009), with the exception of the S. invicta VgR, which also has a four- and eight-repeat arrangement of the LDLRA domains (Chen et al., 2004). The arrangement found in ticks is also different from that found in classical LDLRs, which have a single, seven-repeat domain (Yamamoto et al., 1986), or vertebrate VgRs (Bujo et al., 1994; Okabayashi et al., 1996) and VLDLRs (Takahashi et al., 1992; Sakai et al., 1994), both of which have a single eight-repeat domain. Each LDLRA repeat is approximately 40 aa long and contains six conserved cysteine residues that form a specific disulphide bond pattern (CIeCIII, CIIeCV, and CIVe CVI) that are required for proper folding and binding of the receptor to its ligand (Goldstein and Brown, 1974). The correct folding and disulphide bond formation of the ligand-binding region is dependent on proper coordination of a Ca2þ ion chelated by a cluster of conserved acidic residues (in bold, CDXXXDCXDGSDE) between the fourth and sixth cysteines of each repeat (Atkins et al., 1998; Blacklow and Kim, 1996; Fass et al., 1997). The distribution of EGF-like and YWXD repeats between LBDs in AhVgR is identical to that seen in DvVgR (Fig. 1). Interestingly, in HlVgR, only seven EGF-like repeats are present, there being only three in the first EGF-precursor domain (Fig. 1). This also differs

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from what is seen in insect VgRs, which generally have four EGFlike repeats in the first domain and three in the second (Tufail and Takeda, 2009). Each EGF-like repeat contains three internal disulphide bonds, though in a different bond pattern from that seen in the LDLRA repeats (CIeCIII, CIIeCIV, and CVeCVI; Tufail and Takeda, 2009). Similarly to what is seen in other VgR and LDLRs, the EGF-like repeats bind calcium, with high affinity, to the receptor (Selander-Sunnerhagen et al., 1992; Rao et al., 1995), which acts to induce a rigid conformation of the EGF-like domain, making it resistant to proteolytic degradation (Periz et al., 2005). In AhVgR, EGF-like repeats flank the b-propeller domains, regions comprising six YWXD motifs, that play a vital role in in ligand release and receptor recycling (Springer, 1998; Jeon et al., 2001). AhVgR, like both DvVgR and HlVgR as well as various insect VgR sequences, possesses three b-propeller domains, unlike vertebrate VgRs and LDLRs, which contain a single b-propeller domain (Tufail and Takeda, 2009). Although an OLSD is present in many insect VgRs, such as those from A. aegypti (Sappington et al., 1996), and the cockroaches P. americana (Tufail and Takeda, 2005), Blattella germanica (Ciudad et al., 2006), and L. maderae (Tufail and Takeda, 2007); it is not present in the VgRs reported from S. invicta (Chen et al., 2004), and Drosophila melanogaster (Schonbaum et al., 1995). Both A. hebraeum and D. variabilis possess a 17 aa stretch that is rich in serine and threonine residues directly N-terminal to the transmembrane domain, which may form an OLSD in these species. This region is much shorter than those found in insects, which are generally w30 residues long, but the proportion of S/T residues is similar despite the size difference: A. hebraeum and D. variabilis each possess a total of five S/T residues in this region (29%), compared to six residues found in P. americana and L. maderae (19%), eight in B. germanica (29%) and nine in A. aegypti (32%). The only other fully sequenced tick VgR, from H. longicornis, also has a 17 residue stretch directly Nterminal to the transmembrane domain that is enriched in S/T residues, possessing a total of four S/T residues (24%), but was not reported to possess an OLSD (Boldbaatar et al., 2008). Experimental verification of protein glycosylation sites can be time and laboratory-intensive processes. Although there are multiple computational approaches to identifying O-linked glycosylation sites, unlike N-linked glycosylation sites, O-linked glycosylation sites lack a consensus sequence indicating a potential glycosylation site (Chen et al., 2010). The exact role of the OLSD is still uncertain, when this region is deleted experimentally, there appears to be no effect on ligand-biding, endocytosis, recycling, or degradation (Davis et al., 1986). However, it has been suggested that the OLSD may be involved in promoting receptor stability on the cell surface or in regulation of the cytoplasmic domain (Rodenburg et al., 2006). Similarly to DvVgR, AhVgR has a 22 amino acid transmembrane domain as well as a C-terminal cytoplasmic domain that contains two LL internalization signals as well as the conserved FXNPXF internalization signal. In terms of both sequence similarity and conserved features that have been described, AhVgR is similar to other tick and insect VgRs and LDLRs. The biological significance of the structural variation observed amongst various arthropod species is still unknown. 4.2. Localization and developmental regulation of the AhVgR message The ecdysteroid 20-hydroxyecdysone (20E), rather than JH, appears to be the main regulator of vitellogenesis in ticks. Injection of 20E into D. variabilis (Thompson et al., 2005; Khalil et al., 2011) and A. hebraeum (Friesen and Kaufman, 2002; Seixas et al., 2008) stimulates production of Vg, whereas injection or topical applications of JH had no effect on vitellogenesis (Friesen and Kaufman,

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2004; Thompson et al., 2005). Moreover, Neese et al. (2000), in a comprehensive study, failed to show the presence of JH or its biosynthesis in D. variabilis and Ornithodoros parkeri. Currently, it is not clear how VgR expression is regulated in ticks. In the mosquito A. aegypti, translation of the VgR begins during previtellogenic development, coinciding with elevated levels of juvenile hormone (JH) and is strongly correlated with ecdysteroid titre (Sappington et al., 1996; Cho and Raikhel, 2001; Cho et al., 2006). The role of ecdysteroids in regulation of the mosquito VgR is supported by the presence of two binding sites for the ecdysoneresponsive early gene proteins, E74 and BR, in the AaVgR gene regulatory region 1.5 kb upstream of the VgR gene (Cho et al., 2006). In addition, the level of their expression is correlated with the ecdysteroid titre in females following the blood meal. Injection of partially-fed D. variabilis females with 20E is sufficient to stimulate uptake of Vg into oocytes, implying that VgR is also expressed at this point and is to some degree synchronized with Vg expression (Thompson et al., 2005; Mitchell et al., 2007). This is not the case in A. hebraeum. Injection of 20E stimulated production of Vg, but did not stimulate Vg uptake into oocytes (Friesen and Kaufman, 2004; Seixas et al., 2008). Only when haemolymph collected from engorged females was co-injected with 20E was a significant increase in ovary weight, oocyte size and ovary Vn content observed, suggesting that a ‘Vg uptake factor’ (VUF) is required to promote the uptake of Vg into the oocyte (Seixas et al., 2008). Unlike parthenogenic H. longicornis, in which VgR is expressed at low levels in larvae, nymphs, unfed and partially fed adult females, in addition to the ovaries of engorged females (Boldbaatar et al., 2008), AhVgR is expressed solely in the ovaries of engorged females (Figs. 3 and 4). This is the same expression pattern seen in D. variabilis (Mitchell et al., 2007). From this, it appears that the VUF is not simply a factor that allows Vg to bind to an already present VgR. Rather, it may be involved in regulation of the expression and/ or translation of AhVgR. It is also possible that, like Vg, AhVgR expression is stimulated by 20E, but VUF is expressed only upon engorgement and is then somehow involved in allowing AhVg to bind the receptor for uptake.

4.3. The effects of RNA interference on vitellogenesis RNA interference is a powerful tool for studying gene function and disrupting gene expression in ticks (de la Fuente et al., 2007; Smith et al., 2009). A fair degree of gene knock-down was achieved here, resulting in significantly decreased oocyte length, delayed ovarian development, and a longer latency to oviposition, demonstrating the importance of AhVgR in transport of Vg into developing oocytes. Unlike in D. variabilis or H. longicornis, however, complete abolition of yolk uptake was not observed in VgR-dsRNA injected females, in spite of using four times the amount of dsRNA construct than was used in the D. variabilis experiments (Mitchell et al., 2007). Following engorgement, A. hebraeum requires about 6e7 days to fully develop its ovary (OGP 4), and 10e11 days to begin oviposition under our colony conditions (26  C). It is possible that during this developmental period, the tick produces sufficient AhVgR transcript to overcome the effects of silencing, resulting in a developmental delay as opposed to complete abolition of expression. Although it is possible that there could be a second VgR, or another lipoprotein receptor able to transport Vg into the oocyte, this seems unlikely for two reasons. (1) Only one VgR has been found in both D. variabilis (Mitchell et al., 2007) and H. longicornis (Boldbaatar et al., 2008). (2) In a small number of AhVgR-dsRNA injected ticks, a high degree of knock-down of AhVgR was observed, resulting in no yolk uptake into the oocytes.

4.4. Conclusions This work further adds to our understanding of vitellogenesis in A. hebraeum. We have determined the full-length sequence of AhVgR, and demonstrated that it displays the characteristic features of a vitellogenin receptor. Regulation of vitellogenesis in A. hebraeum is still poorly understood. It is apparent that 20E is insufficient to stimulate yolk uptake by itself, and that a VUF is also needed. The VUF may act to regulate expression and/or translation of AhVgR, or may somehow mediate binding of AhVg to AhVgR. We have shown that unlike in H. longicornis, whose VgR is expressed in larvae, nymphs, partially-fed and engorged females, AhVgR is expressed specifically, and only, in oocytes from ovaries of mated and engorged females. This is also the case in D. variabilis, despite the apparent differences in regulation of yolk uptake. It is still unclear as to how VUF and AhVgR interact to regulate yolk uptake. However, due to the timing of AhVgR expression, we conclude that it does not act to allow binding to a receptor already produced in partially fed females. Additional work is required to determine whether 20E or VUF is responsible for upregulation of AhVgR expression, and if 20E, what role VUF plays in permitting yolk uptake. Acknowledgements This research was generously funded by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to WRK. We are most grateful to Drs. DeMar Taylor and Mari Ogihara (née Horigane) for their invaluable technical advice regarding in situ hybridizations. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389e3402. Atkins, A.R., Brereton, I.M., Kroon, P.A., Lee, H.T., Smith, R., 1998. Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor. Biochemistry 37, 1662e1670. Blacklow, S.C., Kim, P.S., 1996. Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL receptor. Nat. Struct. Biol. 3, 758e762. Boldbaatar, D., Battsetseg, B., Matsuo, T., Hatta, T., Umemiya-Shirafuji, R., Xuan, X., Fujisaki, K., 2008. Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission of Babesia parasite. Biochem. Cell Biol. 86, 331e344. Bujo, H., Hermann, M., Kaderli, M.O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., Schneider, W.J., 1994. Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family. EMBO J. 13, 5165e5175. Chen, M.E., Lewis, D.K., Keeley, L.L., Pietrantonio, P.V., 2004. cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Insect Mol. Biol. 13, 195e204. Chen, S.A., Lee, T.Y., Ou, Y.Y., 2010. Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and nontransmembrane proteins. BMC Bioinform. 11, 536. Cho, K.H., Cheon, H.M., Kokoza, V., Raikhel, A.S., 2006. Regulatory region of the vitellogenin receptor gene sufficient for high-level, germ line cell-specific ovarian expression in transgenic Aedes aegypti mosquitoes. Insect Biochem. Mol. Biol. 36, 273e281. Cho, K.H., Raikhel, A.S., 2001. Organization and developmental expression of the mosquito vitellogenin receptor gene. Insect Mol. Biol. 10, 465e474. Ciudad, L., Piulachs, M.D., Bellés, X., 2006. Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant. FEBS J. 273, 325e335. Combet, C., Blanchet, C., Geourjon, C., Deléage, G., 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147e150. Davis, C.G., Elhammer, A., Russell, D.W., Schneider, W.J., Kornfeld, S., Brown, M.S., Goldstein, J.L., 1986. Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts. J. Biol. Chem. 261, 2828e2838. de la Fuente, J., Kocan, K.M., Almazán, C., Blouin, E.F., 2007. RNA interference for the study and genetic manipulation of ticks. Trends Parasitol. 23, 427e433.

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Molecular characterization of the vitellogenin receptor from the tick, Amblyomma hebraeum (Acari: Ixodidae).

We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are mem...
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