Suppressive subtractive hybridization analysis of Rhipicephalus (Boophilus) microplus larval and adult transcript expression during attachment and feeding.

Abstract

Ticks, as blood-feeding ectoparasites, affect their hosts both directly and as vectors of viral, bacterial and protozoal diseases. The tick's mode of feeding means it must maintain intimate contact with the host in the face of host defensive responses for a prolonged time. The parasite-host interactions are characterized by the host response and parasite counter-response which result in a highly complex biological system that is barely understood. We conducted transcriptomic analyses utilizing suppressive subtractive hybridization (SSH) to identify transcripts associated with host attachment and feeding of larval, adult female and adult male ticks. Five SSH libraries resulted in 511 clones (assembled into 36 contigs and 90 singletons) from differentially expressed transcripts isolated from unattached frustrated larvae (95), feeding larvae (159), unattached frustrated adult female ticks (68), feeding adult female ticks (95) and male adult ticks (94 clones). Unattached 'frustrated' ticks were held in fabric bags affixed to cattle for up to 24h to identify genes up-regulated prior to host penetration. Sequence analysis was based on BLAST, Panther, KOG and domain (CDD) analyses to assign functional groups for proteins including: cuticle proteins, enzymes (ATPases), ligand binding (histamine binding), molecular chaperone (prefoldin), nucleic acid binding (ribosomal proteins), putative salivary proteins, serine proteases, stress response (heat shock, glycine rich) and transporters. An additional 63% of all contigs and singletons were novel R. microplus transcripts or predicted proteins of unknown function. Expression was confirmed using quantitative real time PCR analysis of selected transcripts. This is the first comprehensive analysis of the R. microplus transcriptome from multiple stages of ticks and assists to elucidate the molecular events during tick attachment and development.

Full text

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Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during
attachment and feeding
Ala E. Lew-Tabora,b,c,*, Paula M. Moolhuijzena,c, Megan E. Vancea,b, Sebastian Kurscheida,c,
Manuel Rodriguez Vallea,b, Sandra Jarretta,b, Catherine M. Minchina,b, Louise A. Jacksona,b,
Nick N. Jonssona,d, Matthew I. Bellgarda,c, Felix D. Guerreroa,e
aCooperative Research Centre for Beef Genetic Technologies, Australia
bDepartment of Primary Industries & Fisheries; Locked Mail Bag No. 4, Moorooka, Queensland 4105, Australia
cCentre for Comparative Genomics, Murdoch University, South St., WA 6150, Australia
dThe University of Queensland, School of Veterinary Science, Queensland 4072, Australia
eUS Department of Agriculture, Agricultural Research Service, 2700 Fredericksburg Rd., Kerrville, TX 78028, USA
1. Introduction
The cattle tick, Rhipicephalus (Boophilus) microplus, is
one of the most economically important ticks affecting the
global cattle population (McCosker, 1979). Currently, R.
microplus and its associated pathogens which can be
transmitted to cattle can lead to severe agricultural losses
Veterinary Parasitology xxx (2009) xxx–xxx
A R T I C L E I N F O
Keywords:
Cattle-arthropoda
Rhipicephalus (Boophilus) microplus
Larvae
RNA
Gene expression
Subtractive hybridization
A B S T R A C T
Ticks, as blood-feeding ectoparasites, affecttheir hosts both directly and asvectors of viral,
bacterial and protozoal diseases. The tick’s mode of feeding means it must maintain
intimate contact with the host in the face of host defensive responses for a prolonged time.
The parasite–host interactions are characterized by the host response and parasite
counter-response which result in a highly complex biological system that is barely
understood. We conducted transcriptomic analyses utilizing suppressive subtractive
hybridization (SSH) to identify transcripts associated with host attachment and feeding of
larval, adult female and adult male ticks. Five SSH libraries resulted in 511 clones
(assembled into 36 contigs and 90 singletons) from differentially expressed transcripts
isolated from unattached frustrated larvae (95), feeding larvae (159), unattached
frustrated adult female ticks (68), feeding adult female ticks (95) and male adult ticks
(94 clones). Unattached ‘frustrated’ ticks were held in fabric bags affixed to cattle for up to
24 h to identify genes up-regulated prior to host penetration. Sequence analysis was based
on BLAST, Panther, KOG and domain (CDD) analyses to assign functional groups for
proteins including: cuticle proteins, enzymes (ATPases), ligand binding (histamine
binding), molecular chaperone (prefoldin), nucleic acid binding (ribosomal proteins),
putative salivary proteins, serine proteases, stress response (heat shock, glycine rich) and
transporters. An additional 63% of all contigs and singletons were novel R. microplus
transcripts or predicted proteins of unknown function. Expression was confirmed using
quantitative real time PCR analysis of selected transcripts. This is the first comprehensive
analysis of the R. microplus transcriptome from multiple stages of ticks and assists to
elucidate the molecular events during tick attachment and development.
Crown Copyright ? 2009 Published by Elsevier B.V. All rights reserved.
* Corresponding author at: Department of Primary Industries &
Fisheries; Locked Mail Bag No. 4, Moorooka, Queensland 4105, Australia.
Tel.: +61 7 3362 9502; fax: +61 7 3362 9429.
E-mail address: ala.lew@dpi.qld.gov.au (A.E. Lew-Tabor).
G Model
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Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
0304-4017/$ – see front matter. Crown Copyright ? 2009 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2009.09.033

Page 2

in milk and beef production and restrict the movement of
livestock. The most affected regions of the world are
tropical and sub-tropical countries including northern
Australia, Mexico, South America and South Africa, with
threats to USA cattle populations at southern borders with
Mexico (George et al., 2002). Treatment with acaricide is
the primary means used to control cattle tick infestations,
however resistance to acaricide families is rapidly devel-
oping (Li et al., 2003; Miller et al., 1999, 2005). Currently
resources for tick research are increasing, with the
availability of a global EST library for R. microplus (Wang
et al., 2007) and the current Ixodes scapularis genome
sequencing project (Hill and Wikel, 2005). These resources
will provide the basis for increased cattle tick research
activities, thereby increasing our knowledge of the biology
of ticks and host parasitism.
Molecular events associated with attachment and
feeding have been studied in a number of hard and soft
tick species (Mans et al., 2008; Mulenga et al., 2007a;
Ribeiro et al., 2006). The main basis of these studies has
been to elucidate the parasite: host relationship by
improvedunderstandingofticksalivaryglandcomponents
and how the tick’s secretion of these components is able to
manipulatethehost’sdefensesystems.Thisunderstanding
of the processes which ticks use to adapt to the blood-
feeding environment can greatly facilitate the discovery of
new tick control methods. Limited study of the R. microplus
tick–host interaction has been reported using approaches
applied to other tick species including cDNA library
analysis of salivary gland ESTs in Argas monolakensis
(Mans et al., 2008), Amblyomma cajennense (Batista et al.,
2008), I. scapularis (Ribeiro et al., 2006), Ixodes ricinis
(Chmelar et al., 2008), Ixodes pacificus (Francischetti et al.,
2005) Dermacentor andersoni (Alarcon-Chaidez et al.,
2007); Ornithodoros parkeri (Francischetti et al., 2008b);
differential display analysis of male tick salivary glands
(Amblyomma americanum, D. andersoni) (Bior et al., 2002;
Anyomiet al., 2006); and proteomic analyses of femaletick
sialome of A. monolakensis (Mans et al., 2008) and O.
coriaceus (Francischetti et al., 2008a). One study has
utilized suppressive subtractive hybridization to isolate
transcripts expressed by Amblyomma female ticks which
are up-regulated during ‘host finding’ or pre-attachment
(Mulenga et al., 2007a). Although R. microplus morpholo-
gical studies have been undertaken (Nunes et al., 2006b;
Saito et al., 2005), there are no reports which investigate
themolecularbasisoffeedingorattachmentofR.microplus
ticks and furthermore, no studies to date have attempted
to isolate differentially expressed sequences from tick
larval stages.
This study utilizes a suppressive subtractive hybridiza-
tion technique to isolate 511 sequences up-regulated by
feeding larvae, adult female and adult male ticks and by
adult female and larval ticks responding to host stimuli.
2. Materials and methods
2.1. Ticks and animal sampling
On Day 1, a tick naı ¨ve Hereford female (?9 months age)
was infested with 1.5g (?30,000) N strain larvae (Stewart
et al., 1982) using a tick collar which remained on the
animal while kept in a moat pen (DPI&F Animal Ethics
approval SA2006/03/96). On Day 2, approximately 1000
larvae were placed into a 4 cm2mesh bag and attached to
the neck of animal for 24h in order for the larvae to ‘sense’
host stimuli while also in the presence of other attached
ticks. These ‘frustrated’ larvae were subsequently frozen in
liquid nitrogen or collected into RNAlater RNA Stabiliza-
tion Reagent (Ambion Inc., TX, USA) prior to frozen storage
and extraction, respectively. At 24h (Day 3) approximately
100 attached larvae (feeding larvae) were collected and
stored in RNAlater for RNA extraction. At Day 10, 100
nymphal ticks were collected. Similarly, at Day 17 adult
females were carefully collected and placed into mesh bag
attached to the neck for 24 h prior to harvesting and RNA
extraction (‘frustrated’ females). On Day 17, 40 adult male
ticks (collected from the underside of feeding females) and
50 semi-engorged adult female ticks were collected and
storedinRNAlaterforsubsequentRNAextraction.Skinand
blood were collected from the Hereford to provide host
material for subsequent experiments. For skin tissue
removal, 5 ml lignocaine 2% was injected subcutaneously
to form a line block in the middle of the left side of the neck
with a 25-G needle. Immediately adjacent and distal to one
side, a 5 mm biopsy punch was used to take one full
thickness skin biopsy. The skin was sprayed with disin-
fectant containing fly repellent. Blood was collected from
the tail vein. Tissue was collected from the animal on Days
3, 10 and 17. At Day 21, engorged adult ticks were
collected. On Day 22 the Hereford was treated with
Ivermectin to eliminate remaining ticks at the conclusion
of tick collection.
2.2. Total RNA extraction
RNA was prepared from the whole larvae (frustrated
and feeding), nymph, adult male (feeding/mating stages)
and adult female (frustrated and feeding) ticks collected as
described above. The ticks were ground in liquid nitrogen
using a sterile mortar and pestle prior to processing using
the Qiagen Rneasy kit (QIAGEN CA, USA). RNA was also
prepared from 4000 unattached ‘resting larvae’ and gut,
salivary gland and ovary tissue dissected from 20 adult
female ticks (semi-engorged at 17 days post-infestation).
Samples were used for suppressive subtractive hybridiza-
tion (SSH) and/or qRT-PCR analysis as described below.
2.3. Suppressive subtractive hybridization
SSH was undertaken using the Clontech PCR-SelectTM
cDNA SubtractionKit using cDNAprepared using theSuper
SMARTTMPCR cDNA Synthesis Kit as per manufacturer’s
instructions (Clontech, CA, USA). In order to isolate specific
up-regulated sequences in the following ‘tester’ samples,
cDNA from a mix of up to three ‘driver’ cDNA sequences
were included in each subtraction experiment mixed 1:1
with the tester as follows (also summarized in Table 1): (1)
frustrated larvae: mix of control larvae (unfed), feeding
larvae and skin biopsy 1:1:1 proportions of driver cDNA
(Day3); (2)feeding larvae:mixofcontrollarvae, frustrated
larvae and skin biopsy 1:1:1 (Day 3); (3) male ticks: mix of
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
2
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Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

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control larvae, feeding adult females and skin biopsy 1:1:1
(Day 17); (4) frustrated females: mix of control feeding
adult female ticks and skin biopsy 2:1 (Day 17); (5) feeding
females: mix of control larvae, male adult ticks and skin
biopsy 1:1:1 (Day 17).
2.4. Cloning and sequencing
The differentially amplified transcripts were sub-
cloned into TOPO TA-cloning vectors (ONE SHOT chemi-
cally competent cells) following the manufacturer’s
instructions (Invitrogen Corp., CA, USA). Individual E. coli
colonies were grown in 5 ml LB ampicillin broths for 18h
prior to plasmid extraction from 4 ml using the QIAprep
Spin miniprep kit (QIAGEN, MD, USA) and glycerol storage
of the remaining 1 ml of broth culture (?80 8C). Direct
sequencing of plasmid inserts was undertaken using the
BigDye Vers 3.1 technology (Applied Biosystems, CA, USA)
and analyzed on the Applied Biosystems 3130xl Genetic
Analyser at the Griffith University DNA Sequencing Facility
(School of Biomolecular and Biomedical Science, Griffith
University, Qld, Australia). Sequencing reactions were
prepared using M13 and T7primers in96 well plate format
according to the manufacturer’s instructions (Applied
Biosystems, CA, USA). Sequences were visualized, edited
and aligned using Sequencher Vers 4.5 (Gene Codes
Corporation, MI, USA) to remove all vector and adaptor
sequences prior to further analysis.
2.5. Analysis and sequence function prediction
Vector pCR12.1-TOPO1and SSH adaptor sequence
(Clontech, CA, USA) were clipped from library sequences
using cross-match (Ewing et al., 1998). Individual libraries
were assembled using CAP3 (Huang and Madan, 1999).
Clones within a library were aligned to construct tentative
contigs. Differentially expressed sequences were screened
against the following databases on the CCG (Centre for
Comparative Genomics:
Grendel HPC system (Hunter et al., 2005): NCBI protein
(non-redundant and patent) (National Centre for Biotech-
nology Information: http://www.ncbi.nlm.nih.gov), String
v7 database built on Unicellular (COG) and Eukaryotic
Clusters (KOG) of Orthologous Groups (von Mering et al.,
2007), Clusters of Orthologous Groups of proteins COG
(Tatusov et al., 2003), tigr_bmigi.062608 (The Gene Index
Project http://compbio.dfci.harvard.edu/tgi/) (Quacken-
bush et al., 2000) and NCBI Conserved Domain database
(CDD) (Goonesekere and Lee, 2008). All alignments were
http://ccg.murdoch.edu.au/)
conducted using the BLAST program suite (Altschul et al.,
1990) except for the NCBI Conserved Domain data where
RPSBLAST was used (Goonesekere and Lee, 2008). The
alignment results were then summarized using BIOPERL
(Stajich et al., 2002) scripts based on alignment percent
identity (PID), query coverage and an expected value
thresholds, >25%, >75% and <1e-05 respectively. For
categories not found in the COG database Panther (Protein
ANalysis THroughEvolutionary
www.pantherdb.org/panther/goToPanther.jsp) categories
were assigned manually.
Relationships http://
2.6. qRT-PCR analysis
Primers were designed using emboss version 6.0.1
eprimer3 (Rice et al., 2000) set using the following
parameters: -minsize 22, -osize 24, -maxsize 27, -mintm
55, -maxtm 65, -maxpolyx 4, -gcclamp 2, -productsize
100, -mingc 35, -maxgc 65. Primer sets were then
screened against bovine nucleotide sequence using Blastn
(Altschul et al., 1990) with an expected value 100. Primer
alignments were then screened using a custom Bioperl
(Stajich et al., 2002) script for matches forward and
reverse to ensure these sets would not amplify bovine
sequences. Primer sequences, PCR product and annealing
temperaturesforalltargetsarelistedinTable2.cDNAwas
synthesized using SuperscriptTMIII First-Strand Synthesis
System for RT-PCR (Invitrogen Corp., CA, USA) and
duplicate qPCRs (10 ng per reaction) undertaken using
the SensiMix dT kit (Quantace Ltd., Watford, UK) in the
Corbett RotorGene 3000 (QIAGEN/Corbett, Sydney, Aus-
tralia) using the following profile: 95 8C 10 min, 45 cycles
of 95 8C 15 s, 55 or 60 8C 30 s (see Table 2 for optimal
temperatures per assay), 72 8C 30 s, followed by a melt
analysis 72–90 8C 30 s on the first step, 5 s holds for
subsequent steps, according to manufacturer’s instruc-
tions for SYBR green detection. All assays were first
optimised on a cDNA pool consisting of whole adult
female, adult male and larval cDNAs prior to screening
samples prepared from all stages including extracts
prepared specifically from female gut, salivary gland
and ovaries. Assays with the observed consistent ampli-
fication of duplicates on a standard curve (R2> 0.95)
giving efficiency values of 2.0 (within 15%) were
considered acceptable for normalisation and expression
analysis. The expression profiles (average of two reac-
tions) were normalised against the R. microplus actin gene
(Nijhof et al., 2007) using the Mean Normalised Expres-
sion method (Muller et al., 2002).
Table 1
Summary of each SSH library including the total number of clones and corresponding contigs and singletons following assembly.
Library (tester sample)Drivers No. of clonesNo. of contigsNo. of singletons
‘Frustrated’ larvae (L3)
Feeding larvae (L2)
Adult males (M1)
‘Frustrated’ adult females (F2)
Feeding adult females (F1)
Unfed larvae, feeding larvae, bovine skin biopsy
Unfed larvae, frustrated larvae, bovine skin biopsy
Unfed larvae, feeding females, bovine skin biopsy
Feeding females, bovine skin biopsy
Unfed larvae, male ticks, bovine skin biopsy
95
159
94
68
95
7
4
9
5
11
23
3
19
27
18
Totals
Summary
511
511 clones
36
126 contigs and singletons
90
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

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3. Results
3.1. Subtraction library summary
A total of 511 clones were isolated from the 5 libraries
as described below and a summary of the number of
clones, contigs and singletons per library is presented in
Table 1. The 511 clones resulted in 36 consensus contigs
and 90 singletons, 63% of which were novel R. microplus
transcripts or similar to hypothetical proteins in other
species with no known function. Table 3 describes the
protein hits for each of the contigs and singletons using
Panther, KOG and CDD analysis into specific categories.
Twohypothetical protein
(insect) and Brugia malaya (nematode), present in most
libraries were confirmed using Blastn to be homologous
hits,
Thermobiadomestica
Table 2
Primer sequences for qRT-PCR validation of libraries.
Library and contig/cloneProtein identity/GenBank accessionsPrimers (50-30) f: forward; r: reverse Annealing, 8C
Frustrated larvae
Clone 55Glycine rich f TTCGAAGGTTCGCTTTATCC
r GTGGTTATGGCGGCTATGG
55
Clone 15.5 Putative salivary secreted
aGenBank accession to be updated
Contig 2Hypothetical proteobacterial f CCTGTTTCCCATCGACTACG
r ACCTACACGCCGAAAGTCC
60
Clone 21Hypothetical Drosophila f CCACTGCTGACGTCACTCC
r CTGCGGTGAACCTAACATCC
60
Clone 17Unknown f TAGGACTGCCACAATCATCG
r TTCGTCTAAAATGGGACTGC
60
Feeding larvae
Contig 1 Cuticlef GCGACTGCATTATTTCTATTTCCT
r TCGAAGTTAGAAGGTTCACAACAG
60
Contig 1AATPasef TGATTCTCATCGGTCTAAACTCAG
r GACCTCGATGTTGGATTAGGATAC
60
Contig 2
A. hebraeum mitochondrial genes f TGATTCTCATCGGTCTAAACTCAG
r GACCTCGATGTTGGATTAGGATAC
60
Adult male ticks
Clone 63
R. appendiculatus immunodominant salivaf CGTCGGTCTTGTGAACTTCG
r CCAGTACCTCAGCCATACCC
60
Clone 99 RNA polymerasef TTCAGCTCTAGACGCAATCG
r TCTCCGTGTTTTCAACATGC
60
Clone 10 Trypsin-like serine protease
aGenBank accession to be updated
Contig 2Unknownf AGTCTTCATTTTCCGCAACG
r CCATAAATGCGTCAGACACG
60
Contig 5Unknownf CCTGAGGACACCTCTCATCC
r CTGCGATGACCGCTAATACC
60
Frustrated adult female ticks
Clone 54Trypsin-like serine protease
aGenBank accession to be updated
Contig 3Cuticlef CGTTGAGGGTCACATCAGC
r CCGAGGTACTGCCAACTACG
55
Clone 68Unknownf GATCTACGTTTCCTTCAATCATAGG
r GCAACAATTTGATGATACAGTTCG
60
Clone 78 Unknown
aGenBank accession to be updated
Feeding adult female ticks
Contig 9 Female specific tick histamine binding protein-1
aGenBank accession to be updated
Clone 64Hypothetical culex f ACCAGGTGTGACTGCTCTCC
r GGGTATAGGGGCGAAAGACC
55
Contig 6Unknown f ACGGCACCCAAACTAACG
r TTTCTGAACCAGCGGATACC
55
Clone 59 Unknown f ATTTCGCCTTCGAAGATTGC
r CAAGCTTCCTGCTCTGTCG
55
Clone 75 Unknown
aGenBank accession to be updated
aSequences for these primers can be obtained from the corresponding author following patent registration. See Table 4 describing GenBank accessions
for the remainder of the clones listed in this table.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 5

Table 3
Summary of predicted protein hits (Blast-X and CDD analysis) in each library based on Panther and specific categories.
Molecular function
classification
(Panther)
Total
contigs
Tick stage enriched–no. of clones per contigDescription of protein hitsa
Frustrated
larvae
Feeding
larvae
MaleFrustrated
female
Feeding
female
Tick species homologuea
e-ValueNon-tick or domain homology
Cuticle protein22
Ixodes ricinis cuticle protein 10.9
I. ricinis cuticle protein 10.9
1e-32
4e-102
Enzyme319e-42
Nasonia vitripennis ubiquitin-conjugating
enzyme rad 6 [predicted]-Ubiquitin
protein ligase
52
Amblyomma americanum ATPase FoF1 subunit 6
Rhipicephalus sanguineus ATP-synthase F0 subunit 6
6e-8
7e-142
Glycine rich protein23
1
Argas monolakensis GGY domain protein 3e-7
2e-9 Domain pfam07172–glycine rich protein family
Immunodominant
protein
11
R. appendiculatus 20/24 kDa immunodominant
saliva protein
3e-13
Ligand binding13
R. appendiculatus female-specific histamine
binding protein-1
3e-56
Molecular chaperone211e-31
Ornithorhynchus anatinus similar to
T-complex protein 1 subunit epsilon
Nematostella vectensis predicted protein
[domain cd00632, Prefoldin]
1 6e-25
Nucleic acid binding101
1
I. scapularis ribosomal protein L214e-78
2e-54
3e-35
Limulus polyphemus elongation factor-2
Lycosa singoriensis translation elongation
factor-2
Domain PRK11192–ATP dependent
RNA helicase
Domain pfam08208–RNA polymerase A34
5
114e-8 1e-6
1 9e-6
8e-77
4e-7
2e-22
4e-47
1
1
Ornithodoros parkeri 40S ribosomal protein S3
O. parkeri ribosomal protein L19
Haemaphysalis qinghaiensis ribosomal protein L23
I. scapularis ribosomal protein S17
2
1
Putative salivary
protein
31
1
I. scapularis putative salivary secreted protein
I. scapularis putative salivary protein
3e-41
1e-20
1e-92
Culicoides sonorensis putative salivary protein
Thyropin precursor11
O. moubata putative thyropin precursor1e-13
Serine protease21 3e-27
Drosophila pseudoobscura GA17401-PA
predicted [domain cd00190-secreted
trypsin-like serine protease]
Domain cd00190-secreted trypsin-like
serine protease
1 6e-24
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Table 3 (Continued)
Molecular function
classification
(Panther)
Total
contigs
Tick stage enriched–no. of clones per contig Description of protein hitsa
Frustrated
larvae
Feeding
larvae
MaleFrustrated
female
Feeding
female
Tick species homologuea
e-ValueNon-tick or domain homology
Stress response31 1e-37
Opistophthalmus carinatus heat shock
protein 90
Lilium longiflorum putative senescence
associated protein
Cupressus sempervirens putative senescence
associated protein
3 1e-34
64e-35
Toxin12 0.024Domain pfam07740, Toxin 12, Spider
potassium channel inhibitory toxin
Transferase11 1e-22
Tetraodon nigroviridis hypothetical [domain
pfam04101, glycosyltransferase]
Transporter11 1e-50 Domain pfam04752, Ca2+/H+cation
transport-like protein
DNA mitochondrial
and rRNA
sequences
111105a
59a
17a
49a
[aA. hebraeum mitochondrial NADH
dehydrogenase subunit rDNA, AY059171]
[aR. haemaphysaloides 18S rDNA, DQ839552]
<1e-5
aThermobia domestica hypothetical protein
335143
<3e-22
aBrugia malayi hypothetical protein
Hypothetical
proteins
81 7e-10
2e-24
2e-16
2e-4
2e-17
3e-8
3e-12
2e-8
Drosophila erecta
V. vinifera
Uncultured beta proteobacterium
25
3
1
3
H. qinghaiensis
Rattus norvegicus
1
1
1
R. haemaphysaloides
Culex pipiens quinquefasciatus
Tribolium castaneum
Unknowns
Singletons
Contigs/no. clones
70
13
3/6
0
0
13
6/15
1913
3/80
Total clones n = 511 12695 15994 6895
aAlthough hypothetical protein hits are described as T. domestica GenBank CAM36311 and B. malaya XP_001895031, the DNA sequences are highly homologous to tick mitochondria (A. hebraeum) and ribosomal
genes (R. haemaphysaloides 18S rDNA) at e values of <7e-87 and 0 (100% homology), respectively. For the A. hebraeum sequences: feeding larval library consisted of 105 clones which were assembled into 2 contigs
and 3 singletons; adult male library consisted of 2 contigs; frustrated adult female library consisted of 1 contig and 1 singleton; and the feeding adult female library consisted of a single contig.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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Table 4
List of singleton and contig clones with corresponding GenBank accessions (this study).
LibraryContig or clone no.dbEST Id User IdGenbank accession
Feeding femaleClone 59
Clone 64
Contig 6
62562036
62562035
64498247
64498248
F1-2-A_Clone_59
F1-3-A_Clone_64
F1-2-A_Clone_54
F1-2-A_Clone_50
GE650060
GE650059
GO253184
GO253185
Feeding larvaeContig 1 62562129
62562080
62562127
62562128
62562057
62562116
62562076
62562079
62562049
62562077
62562063
62562083
62562081
62562058
62562096
62562055
62562109
62562106
62562059
62562065
62562105
62562132
62562068
62562112
62562066
62562056
62562126
62562073
62562053
62562061
62562100
62562060
62562117
62562084
62562102
62562101
62562099
62562047
62562115
62562110
L2-2-A_clone_11
L2-2-A_Clone_59
L2-3-A_Clone_41
L2-3-A_Clone_10
L2-3-A_Clone_11
L2-3-A_Clone_12
L2-3-A_Clone_13
L2-3-A_Clone_14
L2-3-A_Clone_15
L2-3-A_Clone_18
L2-3-A_Clone_2
L2-3-A_Clone_20
L2-3-A_Clone_21
L2-3-A_Clone_25
L2-3-A_Clone_26
L2-3-A_Clone_27
L2-3-A_Clone_29
L2-3-A_Clone_36
L2-3-A_Clone_37
L2-3-A_Clone_38
L2-3-A_Clone_39
L2-3-A_Clone_4
L2-3-A_Clone_40
L2-3-A_Clone_43
L2-3-A_Clone_46
L2-3-A_Clone_5
L2-3-A_Clone_50
L2-3-A_Clone_53
L2-3-A_Clone_57
L2-3-A_Clone_58
L2-3-A_Clone_59
L2-3-A_Clone_60
L2-3-A_Clone_62
L2-3-A_Clone_65
L2-3-A_Clone_66
L2-3-A_Clone_67
L2-3-A_Clone_7
L2-3-A_Clone_71
L2-3-A_Clone_76
L2-3-A_Clone_77
GE650153
GE650104
GE650151
GE650152
GE650081
GE650140
GE650100
GE650103
GE650073
GE650101
GE650087
GE650107
GE650105
GE650082
GE650120
GE650079
GE650133
GE650130
GE650083
GE650089
GE650129
GE650156
GE650092
GE650136
GE650090
GE650080
GE650150
GE650097
GE650077
GE650085
GE650124
GE650084
GE650141
GE650108
GE650126
GE650125
GE650123
GE650071
GE650139
GE650134
Contig 1A
Feeding larvaeContig 1A62562097
62562054
62562113
62562074
62562120
62562121
62562067
62562130
62562118
62562104
62562088
62562093
62562069
62562125
62562070
62562103
62562071
62562108
62562092
62562135
62562124
62562050
62562094
62562082
L2-3-A_Clone_78
L2-3-A_Clone_79
L2-3-A_Clone_81
L2-3-A_Clone_82
L2-3-A_Clone_85
L2-3-A_Clone_86
L2-3-A_Clone_87
L2-3-A_Clone_89
L2-3-A_Clone_9
L2-3-A_Clone_91
L2-3-A_Clone_93
L2-3-A_Clone_94
L2-3-A_Clone_95
L2-3-A_Clone_96
L2-3-A_clone_1
L2-3-A_Clone_100
L2-3-A_Clone_17
L2-3-A_Clone_19
L2-3-A_Clone_22
L2-3-A_Clone_23
L2-3-A_Clone_24
L2-3-A_Clone_28
L2-3-A_Clone_3
L2-3-A_Clone_31
GE650121
GE650078
GE650137
GE650098
GE650144
GE650145
GE650091
GE650154
GE650142
GE650128
GE650112
GE650117
GE650093
GE650149
GE650094
GE650127
GE650095
GE650132
GE650116
GE650159
GE650148
GE650074
GE650118
GE650106
Contig 2
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(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 8

with the Amblyomma hebraeum mitochondrial region
(containing NADH dehydrogenase subunit 1/ND1 gene,
partial cds-tRNA-Leu gene-16S ribosomal RNA gene,
partial sequence; and ND1 gene, partial sequence), and
Rhipicephalus haemaphysaloides 18S rDNA, respectively.
These DNA clone/contigs hits were retained in the analysis
for each library described below. Quantitative real time
PCR (qRT-PCR) analysis was used to both confirm tester
differential expression and to demonstrate the expression
of selected transcripts across multiple stages and female
tissues (described for each tester/library below). Table 4
summarises corresponding GenBank accessions for clones
and contigs screened in this study.
3.2. Frustrated larvae library
A total of 95 clones were characterized from the
‘frustrated’larvaetesterwithfunctionalproteinsidentified
as a ubiquitin-conjugating enzyme, glycine rich proteins,
ribosomalproteins,thyropin
hypothetical proteins (insect, plant and bacterial) as
summarized in Table 3 and Fig. 1. A further 19 clones
(13 singletons, 3 contigs) did not return any knownprotein
hits or known domains. The majority of transcripts were
represented by 25 clones (single contig) similar to a plant
(Vitis vinifera) hypothetical protein and 35 clones (single
contig) homologous with R. haemaphysaloides 18S rDNA
sequence. qRT-PCR analysis (Fig. 2) confirmed up-regula-
tion of the specific sequences including the glycine rich
protein at 5? higher expression in frustrated larvae and
other stages (nymph 24?, frustrated adult females 2.5?,
feeding adult females 1.55?, salivary gland 1.29?)
compared to the pooled control. A putative salivary
secreted protein up-regulated in larvae and nymphs was
also detected at high levels in female salivary gland tissue
(?5?). Two clones with hypothetical protein hits only
precursor, andthree
Table 4 (Continued)
LibraryContig or clone no. dbEST IdUser Id Genbank accession
62562062
62562095
62562087
62562086
62562072
62562051
62562075
62562123
62562090
62562107
62562098
62562122
62562134
62562078
62562111
62562048
62562089
62562137
62562052
62562119
62562064
62562133
62562131
62562114
L2-3-A_Clone_32
L2-3-A_Clone_33
L2-3-A_Clone_42
L2-3-A_Clone_45
L2-3-A_Clone_47
L2-3-A_Clone_48
L2-3-A_Clone_49
L2-3-A_Clone_52
L2-3-A_Clone_54
L2-3-A_Clone_55
L2-3-A_Clone_56
L2-3-A_Clone_61
L2-3-A_Clone_63
L2-3-A_Clone_64
L2-3-A_Clone_68
L2-3-A_Clone_70
L2-3-A_Clone_72
L2-3-A_Clone_73
L2-3-A_Clone_75
L2-3-A_Clone_83
L2-3-A_Clone_84
L2-3-A_Clone_88
L2-3-A_Clone_90
L2-3-A_Clone_92
GE650086
GE650119
GE650111
GE650110
GE650096
GE650075
GE650099
GE650147
GE650114
GE650131
GE650122
GE650146
GE650158
GE650102
GE650135
GE650072
GE650113
GE650161
GE650076
GE650143
GE650088
GE650157
GE650155
GE650138
Feeding larvaeContig 2 62562085
62562136
L2-3-A_Clone_98
L2-3-A_Clone_99
GE650109
GE650160
Frustrated female Clone 68
Contig 3
62562042
62562043
62562045
F2-3-A_Clone_68.1
F2-3-A_Clone_67.2
F2-3-A_Clone_77
GE650066
GE650067
GE650069
Frustrated larvae Clone 17
Clone 17
Clone 21
Clone 55
Contig 2
62562138
62562138
62562140
62562139
62562144
62562143
62562142
L3-3-A_Clone_17
L3-3-A_Clone_17
L3-3-C_Clone_21
L3-3-A_Clone_55
L3-3-A_Clone_13
L3-3-A_Clone_14
L3-3-A_Clone_19
GE650162
GE650162
GE650164
GE650163
GE650168
GE650167
GE650166
MaleClone 63
Clone 99
Contig 2
62562145
62562157
62562154
62562151
64498250
64498251
64498249
64498252
M1-2-A_Clone_63
M1-2-A_Clone_99
M1-2-A_Clone_26
M1-2-A_Clone_33
M1-2-A_Clone_89
M1-2-A_Clone_58.3
M1-2-A_Clone_49
M1-2-A_Clone_48.4
GE650169
GE650181
GE650178
GE650175
GO253187
GO253188
GO253186
GO253189
Contig 5
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doi:10.1016/j.vetpar.2009.09.033

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(insect and bacterial) were confirmed by qRT-PCR as up-
regulated in frustrated larvae, as well nymphs, adult males
and femalesalivary gland tissue. Aclone (clone17)with no
known protein hit or domain was shown to be highly
expressed in the frustrated larval (18?) and nymphal
stages (7?) with little or no expression detected in the
other samples screened.
3.3. Feeding larvae library
A total of 159 clones were isolated from feeding larvae
which represented cuticle proteins (2 clones, single
contig), ATPase (52 clones, single contig), and A. hebraeum
mitochondrial genes (105 clones: 2 contigs, 3 singletons),
see Fig. 1. Despite repeated attempts to isolate novel
sequences from this library, a large number of the clones
isolated aligned with the DNA sequence from the A.
hebraeum mitochondrial region which includes NADH
dehydrogenase and 16S rDNA. The collection of feeding
larvae (24 h post-infestation) was difficult thus the limited
sample was not incorporated into qRT-PCR analysis as all
materials were used in the preparation of the libraries.
However, themitochondrial
expressed in nymphs (22?), with levels detected also in
frustrated larvae, feeding adult female ticks and salivary
gland tissue (Fig. 2). The cuticle protein sequence was
highly expressed in nymph stages (129?) and feeding
females (8?) but not in unfed or frustrated larval tick
stages (feeding larval samples not available for screening).
Under the qRT-PCR conditions used here (normalisation to
actin) the ATPase was also shown to be highly expressed in
unfed larvae (212?) and nymphs (25?), Fig. 2.
sequencewas highly
3.4. Adult male library
A total of 94 male tester clones were isolated and
characterized including a Rhipicephalus appendiculatus
immunodominant saliva protein, RNA polymerase, pre-
foldin (molecular chaperone), insect serine protease, toxin
domain (2 clones, 1 contig), 28 unknown sequences (6
contigs and 13 singletons) and an abundance of A.
hebraeum mitochondrial sequence clones (n =59 which
assembled into 2 contigs). Fig. 3 summarizes the composi-
tion of the adult male tick library and Fig. 4 demonstrates
qRT-PCR analysis of selected transcripts. All of the qRT-PCR
assays demonstrated male-specific expression (1–4?
higher expression) with nil/poor expression in all other
samples tested.
3.5. Frustrated adult females
Table 3 and Fig. 5 describe the 68 clones isolated from
the ‘frustrated’ adult female tick library which includes
hits with a cuticle protein (2 clones, single contig),
molecular chaperone, 40S and L19 ribosomal proteins,
serine protease, senescence associated protein (6 clones,
single contig), transferase, 2 hypothetical proteins (Hae-
mophysalis qinghaiensis and a rat sequence) and the A.
hebraeum mitochondrial DNA sequence (17 clones assem-
bling into 1 contig plus 1 singleton). An additional 30% of
the transcripts were unknown with no Blast or CDD hits
represented by 19 singletons. Although feeding adult
femaletickswereusedinthedrivermix,3ofthe4qRT-PCR
assays used to validate the results from this library did not
demonstrate higher expression in the ‘frustrated’ female
tester compared with the feeding female tick sample
(Fig. 6). The qRT-PCR assay based on unknown clone 68
showedahigherexpressioninfrustratedadultfemaleticks
as well as nymphs and ovary tissue compared with other
targets. An assay based on unknown clone 78 showed a
large increase in expression in ‘frustrated’ larvae and
feeding adult females at 780 and 324? respectively. In
addition, a sequence containing a trypsin-like serine
protease domain was shown to be highly expressed in
ovary tissue (4?) with nil expression detected in all other
samples.
3.6. Feeding adult females
A total of 95 feeding female tester clones were isolated
and characterized as an ATP synthase (2 clones, single
contig), histamine binding protein (3 clones, single contig),
Fig. 1. Proportion of the number of transcripts up-regulated in frustrated and feeding larval tick stages (large pie chart) with detailed pie of feeding larvae in
small pie chart.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 10

ribosomal L23 protein (2 clone, single contig), putative
salivary protein (2 clones, single contig), heat shock
protein, 3 hypothetical proteins (R. haemaphysaloides,
mosquito, and beetle), 16 unknowns (3 contigs and 13
singletons) and 49 clones (single contig) homologous to
the A. hebraeum mitochondrial DNA sequence. Results are
summarized in Table 3 and Fig. 5. qRT-PCR results are
presented in Fig. 6. Six qRT-PCR assays confirmed the
differential expression of transcripts in feeding females
with exceptionally high expression of the female specific
histamine binding protein (FSHBP) in feeding (695?) and
frustrated adult females (1388?). High expression of the
FSHBP was also noted in nymph (27?) and slightly higher
levels were amplified in female salivary gland tissue
(2.4?). An unknown (contig 6) also demonstrated excep-
tionally high levels of expression during attachment
Fig. 2. qRT-PCR expression profiles (y-axis indicates average fold change of target gene compared to internal qRT-PCR control) of selected differentially
expressed larval sequences screened across different stages and adult female tissues. Frustrated larvae library assays: glycine rich protein, salivary secreted
protein, hypothetical proteobacterium protein, hypothetical Drosophila protein, and unknown clone 17. Feeding larvae library assays: cuticle protein,
ATPase, and A. hebraeum mitochondrial genes. Legend: UF-L: unfed larvae, Fr-L: ‘frustrated’ larvae, N: nymphs, M: males, Fr-F: ‘frustrated’ females, F-F:
feeding females, FG: female gut, FSG: female salivary gland, and FO: female ovary.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 11

Fig. 3. Summary of transcripts up-regulated in adult male ticks.
Fig. 4. qRT-PCR expression profiles (y-axis indicates average fold change of target gene compared to internal qRT-PCR control) of selected differentially
expressed adult male tick sequences screened across different stages and adult female tissues. Assays: immunodominant saliva protein (R. appendiculatus),
RNA polymerase, trypsin-like serine protease, A. hebraeum mitochondrial, and unknown contig 2. Legend: UF-L: unfed larvae, Fr-L: ‘frustrated’ larvae, N:
nymphs, M: males, Fr-F: ‘frustrated’ females, F-F: feeding females, FG: female gut, FSG: female salivary gland, and FO: female ovary.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 12

(‘frustrated’) and feeding of adult female ticks, as well as
nymphalstages and female gut tissue butnot in adult male
or larval stages (Fig. 6). qRT-PCR analysis of the hypothe-
tical mosquito protein sequence was also shown to be
highly expressed in the frustrated larval sample (8?) and
feeding females (5?), with transcript levels also detected
in frustrated females (2?), nymphs (1.4?) and female gut
tissue (1.4?). Assays based on unknown clones 59 and 75
demonstrated high transcript levels in feeding and
frustrated female tick samples.
4. Discussion
This study demonstrates the application of subtractive
suppressive hybridization (SSH) analysis to identify
differentially expressed transcript associated with R.
microplus larval and adult tick attachment and feeding
in response to host stimuli. Out of the five libraries studied,
the feeding larval library did not yield a large variety of
clones, however it is feasible that transcripts associated
with cuticle production, the mitochondria (NADH dehy-
drogenase, 16S rRNA) and energy (ATPase) are most
abundant in this larval stage. Although the A. hebraeum
mitochondrial sequences were identified in most of the
libraries in this study, gene expression analysis confirmed
that library specific transcripts similar to the A. hebraeum
mitochondrial sequence were in fact stage specific. In
addition, approximately 16–28% of clones from all tester
libraries represented novel sequences with no known
protein or domain hits. An additional 8 sequences matched
hypothetical proteins in other species, also with no known
associated function. The difficulty of identifying tick
sequences has been noted in other tick transcriptome
studies (Mulenga et al., 2007a; Ribeiro et al., 2006). Not all
transcripts were confirmedin qRT-PCR analyses with a few
assays preferentially amplifying driver sequences (unfed
larvae and feeding adult female ticks in frustrated larvae
and adult female libraries respectively). This was mainly
attributed to the use of a single gene for qRT-PCR
normalisation as the generally the tester transcripts which
could be identified showed evidence of relevant stage
specificity. Insights into the potential function of specific
sequences identified in the libraries will contribute to the
elucidation of R. microplus attachment and feeding. This is
the first study analyzing the expression of tick larvae in
response to host stimuli (‘frustrated’ larvae) and the first
analysis of R. microplus differentially expressed sequences
isolated using SSH techniques.
Our study demonstrated the differential expression of a
tick ATPase in 32% of the transcripts isolated from the
feeding larval library and a slight up-regulation of this
ATPase in the female salivary gland. We also demonstrated
the up-regulation of an ATP synthase in female ticks, at
higher levels than the larval ATPase (not shown). ATPase
activity has been shown to be localized in the salivary
glands of female R. microplus ticks during the feeding
period (Nunes et al., 2006a) and an abundance of ATP
synthase ESTs were identified in I. ricinis and D. andersoni
femalesalivaryglandstudies(Alarcon-Chaidezetal.,2007;
Chmelar et al., 2008), confirming ATPase activity in unfed
females as well as feeding female stages as demonstrated
in this study. The above studies (Alarcon-Chaidez et al.,
2007; Chmelar et al., 2008; Nunes et al., 2006a) did not
measure ATPase in larval stages but high levels of ATPases
are linked to intensive secretory activity and thus logically
could be expressed by larval stages (Sauer et al., 2000).
A number of housekeeping genes were identified in
each library, which did appear to be stage specific. Each
library (apart from feeding larvae) yielded tick specific
ribosomal protein clones which appeared to be unique for
each particular tick stage. Specific ribosomal proteins have
been identified in male A. americanum and D. andersoni
ticks, and I. ricinis female sialome analyses (Bior et al.,
2002; Chmelar et al., 2008). In addition, elongation factor 2
Fig. 5. Summary of transcripts up-regulated in frustrated and feeding adult female tick stages (large pie chart with details of frustrated female transcripts)
with the smaller pie chart describing feeding female transcripts.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 13

Fig. 6. qRT-PCR expression profiles (y-axis indicates average fold change of target gene compared to internal qRT-PCR control) of selected differentially
expressed female sequences screened across different stages and adult female tissues. Frustrated female library assays: trypsin-like serine protease, cuticle
protein, unknown clone 68, and unknown clone 78. Feeding female library assays: ATP synthase, female-specific histamine binding protein-1, senescence
associated protein, hypothetical Culex spp. protein, unknown clone 59, and unknown clone 75. Legend: UF-L: unfed larvae, Fr-L: ‘frustrated’ larvae, N:
nymphs, M: males, Fr-F: ‘frustrated’ females, F-F: feeding females, FG: female gut, FSG: female salivary gland, and FO: female ovary.
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
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doi:10.1016/j.vetpar.2009.09.033

Page 14

sequences associated with enhanced ribosomal function
which were previously identified in D. andersoni female
salivary gland EST analyses (Alarcon-Chaidez et al., 2007)
were also isolated in the R. microplus feeding female and
frustrated larval libraries in this study. The up-regulation
of ribosomal RNA particularly of mitochondrial origin may
be associated with the increased activity of the mitochon-
dria during certain stages of tick development such as
feeding. It has been demonstrated that growth and
differentiation relies on the mitochondrial respiratory
chainasthemajorsourceofATPinnematodedevelopment
(Tsang and Lemire, 2002) and that tick mitochondrial
NADH dehydrogenase and lipids are produced in feeding
females and salivary gland cells, respectively (Chmelar
et al., 2008; Denardi et al., 2006). A large number of clones
were isolated from feeding larvae that were associated
with mitochondrial sequences (NADH dehydrogenase, 16S
rRNA), thus correlating with the abundance of ATPase
clones possibly associated with the rapid growth phase of
feeding larvae. In contrast, the ‘frustrated’ larval library
consisted of many more diverse transcripts including
glycine rich and GGY proteins, salivary proteins, a putative
cysteine protease inhibitor, ribosomal proteins and a
number of diverse transcripts with no known prediction.
Cuticular protein transcripts were shown to be up-
regulated during the stages of feeding larvae (the same
clone up-regulated to a lesser degree in feeding females),
whereasacuticleproteincloneisolatedfromthefrustrated
female library was up-regulated in nymph and female
stages but not unfed and frustrated larvae (expression in
feeding larval stages could not be confirmed). There are
thus perhaps stage-specific cuticle proteins and indeed the
structure of the cuticle has been shown to change in I.
ricinis during feeding (Dillinger and Kesel, 2002), thus the
concept that the protein could change during development
is feasible. Curiously, recent tick female transcriptomic
studies undertaken in Ixodes, Dermacentor or Amblyomma
ticks did not yield cuticle protein transcripts (Alarcon-
Chaidez et al., 2007; Chmelar et al., 2008; Mulenga et al.,
2007a). Logically however, cuticle proteins were found to
be most abundant in feeding stages but would not
necessarily be found in salivary gland secretions.
The ‘frustrated’ larval library aimed to isolate tran-
scripts differentially expressed by larvae while attempting
to attach to the host. This is the first tick study that targets
larval stages to study the molecular basis of attachment to
the host. Glycine rich proteins were identified confirming
results obtained in a proteomic analysis of unfed R.
microplus tick larvae (Untalan et al., 2005). Other studies
have demonstrated that GGY proteins are mostly asso-
ciated with female salivary gland secretion, tick attach-
ment (Francischetti et al., 2005; Zhou et al., 2006) and
pathogen transmission (Macaluso et al., 2003; Nene et al.,
2004). It is currently not certain what is the function of
these proteins however they possess extracellular matrix
(ECM) domains and have also been reported to be similar
tothecementproteinsutilizedbyticksforhostattachment
and to also putatively have a role in platelet aggregation
inhibition(Guilfoileand Packila, 2004;Ribeiro et al., 2006).
Although both were up-regulated in frustrated larvae, one
was highly expressed in unfed larvae and the other in
nymphet stages as demonstrated by qRT-PCR analysis.
Thus it is feasible, given the abundance in female stages
also in the above reports, that there is a diverse family of
GGY domain and glycine rich proteins perhaps associated
with different stages and functions. This observation was
recently confirmed in a comparative sialomic study of soft
and hard ticks where it was demonstrated that R. microplus
appears to harbour an abundance of Pro/Gly rich protein
genes (n =27) compared to other tick species: Amblyomma
spp., Ixodes spp., D. andersoni and R. appendiculatus which
comprised approximately 0–9 Pro/Gly rich genes only
(Mans et al., 2008).
A cysteine proteinase inhibitor (cystatin) previously
isolated from the R. microplus fat body (engorged females
cDNA library) was thought to assist with yolk processing
during embryogenesis (Lima et al., 2006). The putative
cysteine proteinase inhibitor isolated from the frustrated
larval library here is more similar to the soft tick
Ornithodoros moubata thyropin precursor that has an
identified thyroglobulin Type 1 domain associated with
cysteine protease inhibitors. In studies using RNAi to
inhibit the A. americanum cystatin, tick blood feeding was
subsequently inhibited, thus indicating that cystatins are
associated with feeding success and O. coriaceus sialomic
studies have confirmed that cystatins are secreted by
female salivary glands (Francischetti et al., 2008a; Karim
et al., 2005). It is not certain what the function of the
frustrated larval R. microplus cystatin isolated here
however it is likely to be associated with early attachment
and/or the initiation of the host feeding cascade.
Blood feeding is known to up-regulate proteases of
various types (Sonenshine and Hynes, 2008). In this study,
we report the isolation of two stage-specific putative
trypsin-like serine proteases from male and female ovary
tissue respectively. Expression of either protease was not
evident in any other tick stages or organs tested. Bior et al.
(2002) identified a cysteine protease in Amblyomma and
Dermacentor male ticks and a male salivary gland study did
not identify serine proteases in other tick species (Anyomi
et al., 2006). In general, fewer male specific tick
transcriptome studies have been undertaken and thus it
is possible that male specific serine proteases are
employed by male R. microplus ticks for feeding or other
purposes. In addition, most female tick transcriptome
studies are focused on salivary gland secretion and thus
have not addressed ovary specific gene expression. This
unique transcript arose from our ‘whole’ female tick cDNA
samples which were used as testers in our SSH experi-
ments. Approximately 34–39 ESTs with trypsin-like serine
protease domains have been identified in the BmGI2 R.
microplus EST database (Mans et al., 2008; Wang et al.,
2007), indicating a potential diverse range of function and
stage-specific activities. This abundance of these serine
proteases has been confirmed in transcriptome studies of
female salivary glands from both soft and hard tick species
(Alarcon-Chaidez et al., 2007; Chmelar et al., 2008;
Francischetti et al., 2008b; Mans et al., 2008). In addition,
tick research literature has focused on the activity of serine
protease inhibitors rather than the serine proteases
possibly due to their high abundance and putative role
in host thrombin inhibition (Batista et al., 2008; Mulenga
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
14
G Model
VETPAR-5024; No of Pages 17
Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033

Page 15

et al., 2007b). Further work to characterize these two
exclusively expressed serine proteases will assist to define
their activities in the male tick and the female ovary,
respectively.
Tick histamine binding proteins (HBPs) are associated
with Ixodid tick salivary glands and exhibit ‘‘histamine-
blocking’’ activity (Chinery and Ayitej-Smith, 1977).
Histamine-mediated cutaneous inflammation is one of
the defence reactions host animals mount against blood-
feeding ticks. Histamine is a principal mediator of
inflammatory reactions and is released by the host in
response to tissue damage such as tick feeding. Ixodid ticks
feedontheirhostfor extendedperiodof timeandthusHBP
sequester host histamine at the wound site outcompeting
host histamine receptors thereby overcoming the hosts’
inflammatory responses and enabling successful feeding
(Paesen et al., 1999). Logically and consistently with other
tick species studies, female tick histamine binding protein
was up-regulated in the adult female tick feeding library in
this study. FSHBP knockdown led to a reduced histamine
binding ability in A. americanum salivary gland and an
aberrant tick feeding pattern or host response (Aljamali
et al., 2003). Our adult female libraries also demonstrated
the up-regulation of stress response proteins such as heat
shock protein and ‘senescence associated’ proteins, puta-
tively associated with adaptation to the host environment
as previously demonstrated (Batista et al., 2008; Mulenga
et al., 2007a).
Glycosyl transferase has been shown to be associated at
the end of the hexosamine biosynthetic pathway facil-
itating the protein glycosylation of uridine diphosphate-N-
acetylglucosamine which is also an active precursor of
chitin (Huang et al., 2007). An enzyme up-regulated in our
frustrated female library was demonstrated to have a
glycosyl transferase domain. A recent study by Huang et al.
(2007) demonstrated that this pathway is essential for
Haemaphysalis longicornis stage development, up-regu-
lated with blood feeding but decreasing with engorge-
ment. Our RT assays demonstrated an up-regulation in
salivary gland and ovary tissue. This is consistent with
Huang et al. (2007) study where knockdown of glutami-
ne:fructose-6-phosphate aminotransferase (an enzyme
associated with the start of the hexosamine biosynthetic
pathway)led to the inhibitionof tick bloodfeeding and egg
production.
Frustrated larval and feeding adult female tick libraries
identified clones which matched previously isolated
salivary secreted proteins from Ixodes ticks and insects
(midges).Theresultswere confirmedinnymphandfemale
stages including salivary gland thus confirming salivary
gland secretion. Their abundance however, has been
demonstrated in female salivary gland secretions in many
tick species including D. andersoni, O. parkeri, and Ixodes
spp. (Alarcon-Chaidez et al., 2007; Chmelar et al., 2008;
Francischetti et al., 2008b, 2005; Ribeiro et al., 2006). No
specific function for these hits is known due to the lack of
recognizable protein domains or motifs however a
comprehensive study in I. scapularis has provided pre-
liminary evidence for conservation of particular domains
which are suggesting a role in anti-haemostasis (Fran-
cischetti et al., 2005; Ribeiro et al., 2006). Antibodies
against these I. scapularis proteins demonstrate an inhibi-
tion of tick feeding and pathogen transmission providing
further evidence of the importance of these salivary
proteins (Narasimhan et al., 2007). A number of other
hypothetical and unknown sequences were identified in
our experiments. Expression analysis confirmed the
specificity of some of these unknown sequences to various
stages or organs. Until the completion of several tick
genome sequences and the development of tick specific
annotation resources, the putative functional identifica-
tion of unknown sequences will continue to be limited.
The R. microplus male tick library was enriched using
males collected from the under-side of semi-engorged
female ticks. All male transcripts screened in RT-PCR
assays demonstrated a strong stage specificity of the male
sequences as compared to expression analysis of the larval
and adult female libraries in this study. Apart from
ribosomal proteins, no similarity of the R. microplus
transcripts from our SSH experiments could be identified
in male specific analyses of Amblyomma or Dermacentor
male ticks. Apart from the putative male-specific serine
protease described above, 2 clones with low homology to a
spider toxin domain were identified. It is feasible that the
putative toxin is associated with feeding and thus has anti-
haemostatic activity (Yamazaki and Morita, 2007) or
perhaps the toxin is involved in assisting the male tick
to stabilize movement of the female during mating.
Putative toxins have been identified in female salivary
gland secretions, also with low sequence homology
(Batista et al., 2008; Francischetti et al., 2005; Ribeiro
etal.,2006).Apartfromthe paralysistick,Ixodesholocyclus,
with known toxin production, putative tick
identified in these studies have not been described or
studied in detail. Other adult male specific sequences
included homologues for an ATP dependent RNA helicase,
prefoldin molecular chaperone, cation transport and a R.
appendiculatus immunodominant protein. The adult male
library also had the greatest abundance of novel sequences
compared to the larval and female libraries.
toxins
5. Conclusion
In this study, we investigated the use of the SSH
technique to identify novel transcripts differentially
expressed in attaching (‘frustrated’) and feeding larvae
and adult female ticks, and adult male R. microplus ticks.
Fromatotalof511clones,126contigsandclonesequences
wereanalyzedusingbioinformaticsandgeneexpression.A
high proportion of unknown novel and hypothetical
sequences were identified at 63% of the total contigs
and singletons. The study identified similarities between
genes differentially expressed during attachment and
feeding in larval, nymphal and adult female stages. The
feeding larval library appeared to be mostly associated
with growth and developmental housekeeping genes with
the unattached (‘frustrated’) larvae producing transcripts
also expressed by adult female tick stages (pre-attachment
and feeding). Adult male gene expression resulting from
the SSH clones demonstrated stage-specific expression of
the screened transcripts, with the highest abundance of
unknowns. Novel differentially expressed sequences iden-
A.E. Lew-Tabor et al./Veterinary Parasitology xxx (2009) xxx–xxx
15
G Model
VETPAR-5024; No of Pages 17
Please cite this article in press as: Lew-Tabor, A.E., et al., Suppressive subtractive hybridization analysis of Rhipicephalus
(Boophilus) microplus larval and adult transcript expression during attachment and feeding. Vet. Parasitol. (2009),
doi:10.1016/j.vetpar.2009.09.033