Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 make shallow cement cones, while the metastriate ticks rely on a larger and deeper cement cone possibly to com- pensate their relatively smaller mouth parts [3]. The number of hosts visited by ticks during the parasitic stage of their life cycle also requires adaptations. According to Balashov (1972) [34] and Hoogstraal and Kim (1985) [35] there was a transition from the three host to the two and one host cycle in Hyalomma and in Rhipicephalinae spe- cies of ticks. The biological characteristic of having a sin- gle host is regarded as an adaptation of this immobile ectoparasite to large nomadic animals since ixodid ticks die when they are unable to find a host. Monoxenous ticks are thus better adapted to open environments inhabited by large, grazing ungulates. The ability to molt on the vertebrate reduces the number of necessary encounters and thus increases chances for tick survival. In addition to elucidating the biology of tick salivary proteins, the information contained in this work is rele- vant for the development of vaccines that target GRPs of ticks and that aim for protection against a broad range of species. The approach undertaken in this work can subsi- dize the choice of the different GRPs present in tick sali- vary glands for evaluation as protective antigens. Methods Ticks Adult female ticks of Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense were collected from naturally infested verte- brate hosts; cattle, dogs and horses, respectively. Samples were collected as to cover the feeding process until the phase of rapid engorgement. Ticks of different sizes, but always ≤ 4 mm in body length (before the rapid engorge- ment phase of feeding; approximately three to four days post attachment) were used for salivary gland dissection to avoid degeneration of salivary gland proteins [36,37]. Ticks were collected from a sample of several hosts and over a period of two to five days and, once removed from the hosts, salivary glands were immediately dissected; a total of 20-30 ticks were used per library. Glands were briefly washed in ice-cold 1X PBS and immediately stored in RNA later storage solution at 4°C for 24 hours and (Ambion, Austin, TX, USA) then transferred to -80°C for long term storage. Extraction of mRNA and cDNA library synthesis Poly A+ mRNA from tick salivary glands was isolated using the Micro-Fast Track™ 2.0 mRNA isolation kit (Invitrogen, Carlsbad, California) following the manufac- turer's instructions. mRNA (similar concentrations for all samples) was used to construct the cDNA library using the vector TriplEx2 according to the instructions for the SMART™ cDNA Library Construction kit (Clontech, Palo Alto, California) with some modifications [38] and pack- aged into lambda phage using the Gigapack® III Gold Packaging Extract (Stratagene, La Jolla, California). Page 15 of 17 The phage sample was used as a template for a PCR reaction to randomly amplify cDNAs. The primers used for this reaction were sequences from the TriplEX2 vec- tor. PT2F1 (5' -AAG TAC TCT AGC AAT TGT GAG C- 3') is positioned upstream of the cDNA of interest (5' end), and PT2R1 (5'-CTC TTC GCT ATT ACG CCA GCT G-3') is positioned downstream of the cDNA of interest (3' end). The cleaned PCR product was used as a template for a cycle-sequencing reaction using the Big Dye kit (Applied BioSystems, Foster City, California).The primer used for sequencing, PT2F3 (5'-TCT CGG GAA GCG CGC CAT TGT-3') is upstream of the inserted cDNA and downstream of the primer PT2F1. Sequencing reactions were performed in one direction only on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, California). Bioinformatic tools Detailed description of the bioinformatic treatment of the data can be found elsewhere with some modifications [38,12]. The programs used were written in Visual Basic 6.0 (Microsoft, Redmond, Washington) by one of us (JMR). Briefly the ESTs (raw sequences) were trimmed of primer and vector sequences, clustered into related groups, based on homology, using the BLASTN algo- rithm (minimum identity of 81 nucleotides over 90 nucle- otides) [39], and then assembled and aligned using the CAP3 assembler [40]. The consensus sequences and sin- gletons resulting from the CAP3 assembler were com- pared to the Non-Redundant (NR) protein database of the NCBI; a customized protein database containing all Arachnida sequences available on Genbank, and the Gene Ontology (GO) database [41] using the BLASTX algorithm (downloaded from an executable file obtained from the NCBI FTP site [39]. Since libraries were con- structed in a unidirectional orientation, BLASTX results were only considered if they were on the positive sense strand. A cut-off E-value of 10-3 was considered for anno- tation. All sequences were translated into three Sequences containing >5% non-assigned nucleotides (Ns) or final length of less than 100 nt were removed from the analysis and assumed to be of poor quality. The final out- put was piped into a tab-delimited file imported into an Excel (Microsoft Excel Analysis Tools, Seattle, WA) spreadsheet. We used the χ2 test and Fisher test to analyze differences in the distribution of ESTs in the different libraries. Phylogenetic analysis of glycine-rich contigs was conducted by first aligning sequences obtained from our cDNA library analysis with published GRP sequences recently cataloged by Francischetti et al. (2009) [18] as well as silk protein sequences obtained from Genbank, using ClustalX Sequence Alignment program [42]. Align- ments were manually refined using BioEdit sequence editing software [43]. Phylogeneic associations were determined using neighbour joining (NJ) analysis (Mega