Rhipicephalus Haemaphysaloides by the Suppression Subtractive Hybridization Approach

Journal of Integrative Agriculture 2012, 11(9): 1528-1536 September 2012 RESEARCH ARTICLE

Identification of Differentially Expressed Genes in the Salivary Gand of Rhipicephalus haemaphysaloides by the Suppression Subtractive Hybridization Approach

XIANG Fei-yu, ZHOU Yong-zhi and ZHOU Jin-lin

Key Laboratory of Animal Parasitology, Ministry of Agriculture/Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, P.R.China

Abstract

For the purpose of screening and analyzing the differentially expressed genes from the salivary gland of Rhipicephalus haemaphysaloides, two salivary gland-subtracted cDNA libraries of partially fed female ticks and fed male ticks were constructed using suppression subtractive hybridization (SSH). A total of 247 female expression sequence tags (ESTs) and 168 male ESTs were obtained from the two SSH cDNA libraries. It is predicted that 25 female ESTs and 44 female ESTs contain the 5´ and 3´ ends, respectively, and that 53 male ESTs and 74 male ESTs contain the 5´ and 3´ ends, respectively. To identify the subtraction rate of the two SSH cDNA libraries, the RT-PCR method was used to test 24 female ESTs and 21 male ESTs selected randomly but not repeatedly. The results showed that there were 13 upregulated or differentially expressed genes in the partially fed salivary gland of the female R. haemaphysaloides and that the differentially expressed rate was 54%. In addition, they indicated that there were 9 upregulated or differently expressed genes in the fed salivary gland of the male R. haemaphysaloides and that the differentially expressed rate was 43%. Putative translations of 141 (57%) female ESTs and 125 (74%) male ESTs had similarity to GenBank sequences, and 32 (23%) female ESTs and 29 (23%) male ESTs exhibited similarity to tick proteins, which showed that most of the proteins in the libraries were mainly related to the feeding blood physiology of the ticks.

Key words: Rhipicephalus haemaphysaloides, salivary gland, suppression subtractive hybridization (SSH), cDNA library, expression sequence tag (EST)

Organization 1984). The three-host tick Rhipiceph- INTRODUCTION alus haemaphysaloides is a widely distributed ixodid species in China, India, and other South Asian coun- Ticks rank first as arthropod vectors of fungi, protozoa, tries (Zhou et al. 2006). This tick is a major vector of rickettsiae, bacteria, and viruses, causing diseases in bovine babesiosis in China (Yin et al. 1997) and can non-human vertebrates, and rank second only to mos- also transmit the Kyasanur Forest disease virus, as quitoes as vectors of pathogens to humans (Bior et al. shown by animal experiments (Bhat et al. 1978). The 2002). Tick-borne diseases are particularly devastat- tick-disease complex of R. haemaphysaloides is an im- ing to livestock, especially cattle, with costs estimated portant agriculturally related complex, causing severe to be about $7 billion per annum (Food and Agricultural economic losses in milk and beef production and re-

Received 6 June, 2011 Accepted 10 October, 2011 XIANG Fei-yu, Mobile: 13996999752, E-mail: [email protected]; Correspondence ZHOU Jin-lin, Tel: +86-21-34293411, Fax: +86-21-54081818, E-mail: [email protected]

© 2012, CAAS. All rights reserved. Published by Elsevier Ltd. Identification of Differentially Expressed Genes in the Salivary Gand of Rhipicephalus haemaphysaloides 1529 striction in the traffic of livestock. abundance transcripts and differentially expressed genes Blood-feeding organisms, including ticks, secrete with high subtraction efficiency. For the purpose of bioactive substances that exhibit a range of pharma- screening and analyzing differentially expressed genes cological properties to thwart the host defense mecha- from the salivary gland of R. haemaphysaloides, two nisms in response to attachment (Oaks et al. 1991; salivary gland-subtracted cDNA libraries of female par- Bowman et al. 1997). A large proportion of these tially fed and male fed ticks were constructed using SSH. substances are produced in the salivary glands and secreted into the host during blood feeding. During RESULTS this period, the salivary gland plays a key role that helps the tick feed on blood. The salivary glands of ixodid females undergo remarkable growth and dif- Suppression subtractive hybridization PCR ferentiation during tick feeding as their cells become competent to transport fluid. The mass and protein After 2 rounds of hybridization, the products were am- content of the salivary glands increase about 25-fold, plified by PCR-select with primer pairs and nested primer but the number of cells does not increase (Sauer et al. pairs, and the subtractive cDNA fragments were then 2000). It has been observed that, during the blood obtained. The size and abundance of the PCR products meal, new mRNAs are induced in the salivary glands, were examined by agarose gel electrophoresis. The re- leading to the synthesis of a wide range of new pro- sults showed that the sizes of the subtractive cDNA frag- teins (Leboulle et al. 2000). In contrast to females, ments mainly ranged between 500 and 750 bp (Fig. 1). very little is known about the physiological, biochemical, and molecular changes in the salivary glands of male ticks before or after male attachment to the host. While female ticks imbibe large volumes of blood over 2 wk, male ixodid ticks are intermittent feeders with relatively low blood intake. The mass of the salivary glands of male A. americanum increases about 1.6-fold during feeding (Shipley et al. 1993). The type IV acinus, re- stricted to males, is thought to have a reproductive role, possibly in sperm transfer (Feldman-Muhsam et al. 1970). Wang and Nuttall (1998) provided evidence for male-specific immunoglobulin-G-binding proteins that are hypothesized to be important in protecting the mate during attachment and feeding by nearby females (Wang et al. 1998). It is believed that several these induced genes are essential for the completion of the tick feeding process and the transmission of pathogens. Therefore, to screen the differentially expressed genes of the salivary gland, we can identify the dynamic changes occurring at the bioactive molecular level during male and female tick feeding and obtain valuable functional molecular information. A PCR- select cDNA subtraction method (suppression subtractive hybridization, SSH) was developed by Clontech, USA. Because it includes a nor- Fig. 1 Gel electrophoresis of the subtraction PCR-select products from the salivary gland cDNA of the female (A) and male (B) malization step to equalize the abundance of cDNAs R. haemaphysaloides. Lane 1, subtraction result; lane 2, within the target population, this method can detect low- unsubtraction control; M, DL2000 marker.

Characterization of select clones and sequences regulation, energy metabolism, and antioxidization of the 2 subtractive cDNA libraries (Tables 3 and 4).

After screening the 2 subtractive cDNA libraries with DISCUSSION nested primer 1 and primer 2R, all positive clones in the libraries were sequenced. By searching the primer 1 SSH is a highly efficient method to screen and isolate and primer 2R in the sequences with the software differentially expressed genes. We successfully applied DNAMAN (v4.0), 247 female ESTs and 168 male ESTs SSH in this test and isolated some valuable genes re- were obtained. Based on the characteristic sequence lated to tick feeding. One potential disadvantage of the ACGCGGG and the poly(A) sequence, it was predicted SSH technique first reported by Diatechenko (1996) is that 25 female ESTs and 44 female ESTs would con- the fact that a few micrograms of poly(A) RNA and tain the 5´ and 3´ ends, respectively, and that 53 male some low-redundancy RNA may be difficult to obtain. ESTs and 74 male ESTs would contain the 5´ and 3´ To resolve this problem, an amplification step for both ends, respectively (Table 1). the driver and tester cDNAs was incorporated to generate sufficient quantities of both cDNA samples be- Confirmation of differential expression of genes fore initiating the SSH procedure. In our protocol, we solved this problem with LD-PCR by use of the After the subtracted cDNA library was obtained, it was CLONTECH Super SMART PCR cDNA Synthesis Kit, important to confirm that individual clones did indeed USA. In the typical SSH protocol, the lowest quantity represent differentially expressed genes. This was ac- of RNA is 2 µg. However, for the SMART method, the complished by RT-PCR with randomly selected minimum amount of starting material for standard cDNA sequences. Therefore, 24 female and 21 male se- is 2 ng of the total RNA. In our experiment, the differ- quences were selected randomly but not repeatedly. entially expressed rate was high, and some valuable The primers were designed using DNAMAN (v4.0) genes were isolated. software. The length of all primers was about 20 bp, The present results demonstrate that new gene ex- and all amplified products were about 250 bp, includ- pression occurs in the salivary glands of male and fe- ing the control. The results showed that there were 13 male R. haemaphysaloides. These salivary gland upregulated or differentially expressed genes in the proteins, which play an important role in tick biology, pa r t i a l l y fe d s a l i v a r y gl a nd o f t he fema l e e.g. as histamine binding and immunoglobulin binding R. haemaphysaloides and that the differenti al ly ex- proteins and serine proteinase inhibitors, have been de- pressed rate was 54%. In addition, they indicated that scribed previously (Waxman et al. 1990; Wang et al. there were 9 upregulated or differently expressed genes 1995; Sangamnatdej et al. 2002). Considering the di- in the fed salivary gland of the male R. haemaphysaloides verse roles of putative secreted proteins in blood feeding, and that the differentially expressed rate was 43%. a brief description for each important protein class is

Summary of the results of BLASTx and protein Table 1 Summary of sequences in the salivary gland SSH cDNA homology libraries of R. haemaphysaloides Sex ESTs 5´ end sequences 3´ end sequences Putative translations of 141 (57%) female ESTs and Female 247 25 (10%) 44 (18%) Male 168 53 (32%) 74 (44%) 125 (74%) male ESTs had similarity to GenBank sequences, and 32 (23%) female ESTs and 29 (23%) male ESTs exhibited similarity to tick proteins (Table Table 2 The sequence analysis results of the salivary gland SSH cDNA library of R. haemaphysaloides 2), which showed that most of the proteins in the li- Sex Tick Parasite Others Unknown Annotated rate braries were mainly related to the feeding blood physi- Female 32 (23%) 26 (18%) 83 (59%) 106 (43%) 57% (141/247) ology of ticks, including anticoagulation, immunity Male 29 (23%) 42 (36%) 54 (43%) 43 (26%) 74% (125/168)

Table 3 Summary of the important proteins of the salivary gland SSH cDNA library of the female R. haemaphysaloides Clone Sequence identity gi number Species E value Function F37 Serotonin and histamine binding protein gi|18032205 Dermacentor reticulates 7e-04 Immune modulation F9 Immunosuppressant protein p36 gi|6103605 Dermacentor andersoni 8e-21 Immune modulation F5 Collagen-like protein gi|18309937 Clostridium perfringens 0.008 Helping attach C18 Kunitz-type protease inhibitor 1 gi|50748153 Gallus gallus 3e-04 Anticoagulation C201 Tissue factor pathway inhibitor gi|125934 Oryctolagus cuniculus 7e-10 Anticoagulation F117 ATPase subunit 6 gi|32481105 Rhipicephalus haemaphysaloides 7e-70 Energy metablish F4 Cytochrome b gi|4164569 Rhipicephalus sanguineus 2e-94 Energy metablish F10 Cytochrome oxidase 2 gi|4164559 Rhipicephalus sanguineus 1e-69 Energy metablish C183 NADH dehydrogenase 1 gi|4164564 Rhipicephalus sanguineus 2e-23 Energy metablish C21 Cytochrome oxidase 3 gi|4164562 Rhipicephalus sanguineus 6e-68 Energy metablish F33 Manganese superoxide dismutase gi|33186704 Antrodia camphorata 1e-05 Antioxidzation F81 Catalase gi|984737 Campylobacter jejuni 5e-10 Antioxidzation C29 SocE gi|9652070 Myxococcus xanthus 5e-20 Iron pathway C169 Secreted salivary gland peptide gi|56159947 Ixodes scapularis 2e-08 Unknown F62 Unknown gi|45479213 Rhipicephalus haemaphysaloides 4e-92 Unknown F16 Ribosomal protein S17 gi|24266986 Branchiostoma belcheri 6e-54 Housekeeping gene

Table 4 Summary of the important proteins of the salivary gland SSH cDNA library of the male R. haemaphysaloides Clone Sequence identity gi number Species E value Function M196 Immunoglobulin G binding protein B gi|2352272 Rhipicephalus appendiculatus 4e-79 Immune modulation M199 Secreted histamine binding protein of 22.8 kDa gi|51011532 Ixodes pacificus 0.037 Immune modulation M70 20/24 kDa immunodominant saliva protein gi|28932710 Rhipicephalus appendiculatus 2e-11 Immune modulation M65 36/38 kDa immunodominant saliva protein gi|28932712 Rhipicephalus appendiculatus 0.64 Immune modulation M14 Putative cement protein RIM36 gi|21885262 Rhipicephalus appendiculatus 2e-07 Helping attach M200 von Willebrand factor gi|33285889 Ixodes ricinus 9e-11 Anticoagulation M229 Truncated salivary metalloprotease gi|51011584 Ixodes pacificus 2e-09 Anticoagulation M223 Serine protease gi|7960528 Anopheles gambiae 7e-20 Anticoagulation M151 Cytochrome b gi|4164569 Rhipicephalus sanguineus 3e-96 Energy metablish M168 Cytochrome c oxidase I gi|15808091 Rhipicephalus maculatus 2e-13 Energy metablish M21 Manganese superoxide dismutase gi|33186704 Antrodia camphorata 1e-06 Antioxidzation M204 Catalase gi|984737 Campylobacter jejuni 4e-10 Antioxidzation M159 Vangl2 protein gi|41946856 Homo sapiens 0.010 Signal transduction M171 Cytochrome P450 monooxygenase gi|2995384 Zea mays 5e-16 Detoxification M15 LRRG00134 gi|37654218 Rattus norvegicus 9e-27 - M219 Hematoietic specific protein E3 gi|6685986 Mus musculus 2e-11 Hematoiet M105 Putative transcription factor gi|5531330 Periplaneta americana 8e-15 Transcription presented below. vent the initiation of blood coagulation. Because the major objective of any tick vaccine will be to make it Class 1: The Kunitz domain-containing protein difficult for ticks to obtain a blood meal, tick-encoded anticoagulants should then be considered as potential Proteins of this class either contain a Kunitz domain, as target vaccine antigens. indicated by the SMART algorithms, or are related in sequence similarity to proteins with Kunitz domains. Class 2: The von Willbrand factor One member of this class, gi|15077002, has been char- acterized and shown to inhibit the tissue factor path- Fig. 4 presents one protein sequence that has similarity way of blood clotting by interacting with the extrinsic to the von Willbrand factor. The von Willbrand factor Xase complex, which is a multiprotein complex as- could be predicted to have chymotrypin-elastase inhibitor sembled in a phospholipid surface (Francischetti et al. activity (Nakajima et al. 2005). This protease inhibitor 2002). Sequencing the R. haemaphysaloides cDNA li- possesses a trypsin inhibitor-like domain (TIL) and may braries yielded two proteins containing Kunitz-like target serine proteases (Ribeiro et al. 2006). TIL do- domains. These proteins share sequence similarity to main peptides, which are assumed to regulate host he- two ixolaris salivary proteins (Figs. 2 and 3) that pre- mostatic or inflammatory reactions, have been described

Fig. 2 Sequence alignment of C18 and KTPI (Kunitz-type protease inhibitor 1, gi|50748153). Identical residues are shaded in black. The Kunitz domain is boxed.

Fig. 3 Sequence alignment of C201 and TFPI (tissue factor pathway inhibitor, gi|125934). Identical residues are shaded in black. The Kunitz domain is boxed.

Fig. 4 Sequence alignment of M200 and vWf (von Willebrand factor, gi|33285889). Identical residues are shaded in black. The TIL domain is boxed. in the I. scapularis sialotranscriptome (Valenzuela et al. hemolymph and salivary glands of male and female ixo- 2002). did ticks appear to transport host IgG from the gut back into the host via the saliva (Wang et al. 1994, Class 3: Immunoglobulin G-binding proteins 1999). This host IgG might be damaging to tick tissue. (IGBP) Ticks have developed a mechanism for excreting host IgG via their salivary glands. This mechanism may In the male tick ESTs, one sequence showed high pro- minimize the potential damage caused by the host anti- tein similarity with IGBPB (90%) and IGBPC (52%) of body during feeding, particularly in adult female ticks. Rhipicephalus appendiculatus (Fig. 5). Wang et al. IGBPs in salivary glands then excrete the antibody back (1998) provided evidence for a role of male-specific into the feeding site. Ticks fed on these protein-vacci- IGBP in mate guarding. IGBPs present in the nated hosts died from severe antibody-mediated dam-

© 2012, CAAS. All rights reserved. Published by Elsevier Ltd. Identification of Differentially Expressed Genes in the Salivary Gand of Rhipicephalus haemaphysaloides 1533 age to the gut (Willadsen et al. 1996; Nuttall et al. 2006). 7). This protein shares sequence similarity to the serine Surviving ticks also suffered from a reduction in their protease of another blood-feeding insect, Anopheles engorgement weight and egg-laying capacity. gambiae. Serine proteases are special enzyme families implicated in trypsin, chymotrypsin, thrombin, plasmin, Class 4: Immunosuppressant protein and a tissue plasminogen activator (tPA) (Puente et al. 2003). The latter two enzymes prevent blood Class 6 (Fig. 6) contains one sequence with similarity coagulation. This serine protease of R. haemaphys- to immunosuppressant protein p36, discovered in the aloides may function as an anticoagulant. tick saliva of Dermacentor andersoni. Immunosup- pressant protein is found to modulate T lymphocyte CONCLUSION and macrophage responsiveness by inducing a Th2- type response and inhibiting the production of pro-in- The use of SSH allowed us to identify and isolate dif- flammatory cytokines (Leboulle et al. 2002). This pro- ferentially expressed genes from unfed and fed male tein might play an important role in the modulation of and female R. haemaphysaloides. This method may be host immune responses. Tick saliva contains immuno- useful in identifying factors that are likely to be impor- suppressive factors that allow this blood-feeding ecto- tant in tick feeding and may have significance in the parasite to remain on the hosts and enhance pathogen ability of ticks to transmit pathogens. Novel bioactive transmission (Hannier et al. 2003). substances were obtained from tick salivary glands. These molecules have potential as new anti-tick drug Class 5: Tryp_SPc domain protein target candidates. Furthermore, the analysis of these molecules could provide some information for the de- Sequencing the male R. haemaphysaloides cDNA library veloping of new therapeutic drugs, for example, anti- yielded a protein containing a Tryp_SPc domain (Fig. coagulants for thrombosis. Finally, defining the most

Fig. 5 Sequence alignment of M196, IGBPB (gi|2352272) and IGBPC (gi|2352274). Identical residues are shaded in black.

Fig. 6 Sequence alignment of F9 and p36 (immunosuppressant protein p36, gi|6103605). Identical residues are shaded in black.

Fig. 7 Sequence alignment of M223 and SP (serine protease, gi|7960528). Identical residues are shaded in black. The Tryp_SPc domain is boxed. abundant antigens that may effectively help ticks feed R. haemaphysaloides was used for double-strand cDNA could be an effective approach to developing protec- synthesis. cDNAs were then amplified by LD-PCR, and PCR products were purified with a spin column. tive vaccines directed toward tick salivary molecules.

Suppression subtractive hybridization MATERIALS AND METHODS Analysis of differentially expressed genes was performed Ticks and salivary gland collection by suppression subtractive hybridization using the CLONTECH PCR-SelectTM cDNA Subtraction Kit (Clontech, USA). The full-length cDNA was digested with Rsa I. The The tick R. haemaphysaloides has been maintained by digested cDNA was split into two classes and ligated to feeding on rabbits for several generations in Shanghai Vet- either adaptor 1 or adaptor 2R. Subtractive hybridization erinary Research Institute, Chinese Academy of Agricul- was performed by annealing an excess of the salivary gland tural Sciences. Shanghai, China (Zhou et al. 2003). For cDNAs of unfed female or male R. haemaphysaloides (as a salivary gland collection, adult R. haemaphysaloides were driver) with each sample of the adaptor-ligated tester infested on the ears of a rabbit. Ticks were recovered from cDNAs (the salivary gland of a fed female or male the rabbit after 7 d to female ticks and 15 d to male ticks, R. haemaphysaloides). The cDNAs were heat-denatured and the salivary glands were immediately dissected under and incubated at 68°C for 8 h. After the first hybridization, the microscope. In addition, the salivary glands of the the two samples were mixed together and hybridized again unfed male and female ticks were dissected. The sample with freshly denatured driver cDNAs for 20 h at 68°C. The materials were stored at -80°C until use. two rounds of hybridization generated a normalized population of tester-specific cDNA with different adaptors on RNA extraction each end. After filling in the end, two rounds of PCR amplification were performed to enrich desired cDNAs containing both adaptors by exponential amplification of these Total RNA was extracted from the unfed and fed salivary products. The optimized cycles for the first and second glands of male and female R. haemaphysaloides using PCRs were 30 and 20, respectively, to increase representa- TRIzol Reagent (Invitrogen, The Netherlands) according tion and reduce redundancy of the subtracted cDNA to the manufacturer’s protocol. libraries. cDNA synthesis and amplification by long Constructing the subtracted cDNA libraries distance PCR (LD-PCR) Products from the second PCR were T-A-cloned into a To generate sufficient cDNAs for PCR-select subtraction, vector of pGEM-T-easy. The ligation products were trans- the CLONTECH Super SMART PCR cDNA Synthesis Kit formed into DH5a-competent cells. The transformed cells (Clontech, USA) was applied. 20 ng of mRNA from unfed were plated on LB plates containing ampicillin, X-Gal, and a n d f ed s a l i v a r y g l a n d s o f m a l e a n d f e m a l e IPTG, which allowed for color selection of the colonies.

Finally, all white colonies were selected and further con- Francischetti I M, Valenzuela J G, Andersen J F, Mather T firmed using nested PCR with primer 1 and primer 2R. N, Ribeiro J M. 2002. Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X Sequencing and BLAST homology search and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex. Blood, 99, 3602-3612. All positive clones from the subtracted cDNA library were Food and Agricultural Organization. 1984. Ticks and Tick- selected for sequencing. Sequencing was performed us- borne Disease Control: A Practical Field Manual. vol. ing a T7 or SP6 primer (Genecore Biotech, China). The 1. Food and Agricultural Organization, Rome. pp. iv-xi. BLASTx program was used to search for the cDNA se- Hannier S, Liversidge J, Sternberg J M, Bowman A S. 2003. quence homology of isolated clones in GenBank. The do- Ixodes ricinus tick salivary gland extract inhibits IL-10 main and alignment were analyzed with Genetyx (v4.0) soft- secretion and CD69 expression by mitogen-stimulated ware and SMART algorithms. murine splenocytes and induces hyporesponsiveness in B lymphocytes. Parasite Immunology, 25, 27-37. Validation of differential expression of genes by Leboulle G, Crippa M, Decrem Y, Mejri N, Brossard M, Bollen A, Godfroid E. 2002. Characterization of a novel reverse transcription PCR (RT-PCR) salivary immunosuppressive protein from Ixodes ricinus ticks. The Journal of Biological Chemistry, The differentially expressed genes selected randomly from 277, 10083-10089. the ESTs were further validated by RT-PCR using gene- Leboulle G, Rochez C, Louahed J, Ruti B, Brossard M, specific primer pairs designed by DNAMAN (v4.0). Fed Bollen A, Godfroid E. 2000. Isolation of Ixodes ricinus and unfed salivary gland cDNAs of male and female salivary gland mRNA encoding factors induced during R. haemaphysaloides were amplified by PCR controlled blood feeding. American Journal of Tropical Medicine with the tubulin gene. The quality and specificity of the and Hygiene, 66, 225-233. amplified products were confirmed by visualization on a Nakajima C, da Silva Vaz I J, Imamura S, Konnai S, Ohashi 1% agarose gel. K, Onuma M. 2005. Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland. The Journal of Veterinary Acknowledgements Medical Science, 67, 1127-1131. This work was supported by the National Natural Science Nuttall P A, Trimnell A R, Kazimirova M, Labuda M. 2006. Foundation of China (31172095). Exposed and concealed antigens as vaccine targets for controlling ticks and tick-borne diseases. Parasite References Immunology, 28, 155-163. Oaks J F, McSwain J L, Bantle J A, Essenberg R C, Sauer J Bhat H R, Naik S V, Ilkal M A, Banerjee K. 1978. R. 1991. Putative new expression of genes in ixodid tick Transmission of Kyasanur Forest disease virus by salivary gland development during feeding. Journal of Rhipicephalus haemaphysaloides ticks. Acta Virology, Parasitology, 77, 378-383. 22, 241-244. Bior A D, Essenberg R C, Sauer J R. 2002. Comparison of Puente X S, Sanchez L M, Overall C M, Lopez-Otin C. 2003. differentially expressed genes in the salivary glands of Human and mouse proteases: a comparative genomic male ticks, Amblyomma americanum and Dermacentor approach. Nature Reviews Genetics, 4, 544-558. andersoni. Insect Biochemisty and Molecular Biology, Ribeiro J M, Alarcon-Chaidez F, Francischetti I M, Mans B 32, 645-655. J, Mather T N, Valenzuela J G,Wikel S K. 2006. An Bowman A S, Coons L B, Needham G R, Sauer J R. 1997. annotated catalog of salivary gland transcripts from Tick saliva: recent advances and implications for vector Ixodes scapularis ticks. Insect Biochemistry and competence. Medical and Veterinary Entomology, 11, Molecular Biology, 36, 111-129. 277-285. Sangamnatdej S, Paesen G C, Slovak M, Nuttall P A. 2002. Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam A high affinity serotonin- and histamine-binding F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, lipocalin from tick saliva. Insect Molecular Biology, Sverdlov E D, et al. 1996. Suppression subtractive 11, 79-86. hybridization: a method for generating differentially Sauer J R, Essenberg R C, Bowman A S. 2000. Salivary regulated or tissue-specific cDNA probes and libraries. glands in ixodid ticks:control and mechanism of Proceedings of the National Academy of Sciences of secretion. Journal of Insect Physiology, 46, 1069-1078. the United States of America, 93, 6025-6030. Shipley M, Dillwith J, Bowman A, Essenberg R, Sauer J. Feldman-Muhsam B, Borut S, Saliternik-Givant S. 1970. 1993. Changes in lipids from the salivary glands of the Salivary secretion of the male tick during copulation. lone star tick, Amblyomma americanum, during feeding. Journal of Insect Physiology, 16, 1945-1949. Journal of Parasitology, 79, 834-842.

Valenzuela J G, Francischetti I M, Pham V M, Garfield M K, ticks help their mates to feed. Nature, 391, 753-754. Mather T N, Ribeiro J M. 2002. Exploring the sialome of Waxman L, Smith D E, Arcuri K E, Vlasuk G P. 1990. Tick the tick, Ixodes scapularis. Journal of Experimental anticoagulant peptide (TAP) is a novel inhibitor of blood Biology, 205, 2843-2864. coagulation factor Xa. Science, 248, 593-596. Valenzuela J G, Francischetti I M, Pham V M, Garfield M K, Willadsen P, Smith D, Cobon G, McKenna R V. 1996. Mather T N, Ribeiro J M. 2002. Exploring the sialome of Comparative vaccination of cattle against Boophilus the tick, Ixodes scapularis. Journal of Experimental microplus with recombinant antigen Bm86 alone or in Biology, 205, 2843-2864. combination with recombinant Bm91. Parasite Wang H, Nuttall P A. 1994. Excretion of host immunoglobulin Immunology, 18, 241-246. in tick saliva and detection of IgG-binding proteins in Yin H, Lu W, Luo J. 1997. Babesiosis in China. Tropical tick haemolymph and salivary glands. Parasitology, Animal Health and Production, 29(Suppl. 4), 11S-15S. 109, 525-530. Zhou J, Gong H, Zhou Y, Xuan X, Fujisaki K. 2006. Wang H, Nuttall P A. 1995. Immunoglobulin G binding Identification of a glycine-rich protein from the tick proteins in the ixodid ticks, Rhipicephalus Rhipicephalus haemaphysaloides and evaluation of appendiculatus, Amblyomma variegatum and Ixodes its vaccine potential against tick feeding. Parasitology hexagonus. Parasitology, 111, 161-165. Research, 100, 77-84. Wang H, Nuttall P A. 1999. Immunoglobulin-binding Zhou Y Z, Zhou J L, Cao J, Gong H Y. 2003. A laboratory proteins in ticks: new target for vaccine development method for the maintaining and feeding of four ixodid against a blood-feeding parasite. Cellular and ticks and observation on their partial biology characters. Molecular Life Sciences, 56, 286-295. Chinese Journal of Veterinary Parasitology, 11, 25- Wang H, Paesan G C, Nuttall P A, Barbour A G. 1998. Male 27.

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