Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 num, D. andersoni, R. microplus and R. appendiculatus (Sup-Table 1 of Francischetti et al., 2009). We aligned all sequences similar to silk-like proteins from our libraries and the NR database from Sup-Table 1 (describe in Francischetti et al., 2009) and using the neighbor joining analysis produced the phylogram shown in Figure 4. Sequences generated by the present work are symbolized with a circle ( : R. microplus), a square ( : R. sanquineus ) and a triangule (▲: A. cajennense). In addi- tion to our sequences, 45 other transcript sequences of other species ticks are represented: Dermacentor ander- soni and R. appendiculatus, both Brevirostrata, heteroxe- nous ticks, and A. variegatum and A. americanum, Longirostrata, heteroxenous ticks. Sequences of R. microplus from other sources were also included in the analysis. To the best of our knowledge there are no repre- sentatives of Longirostrata, monoxenous ticks that could be included in this analysis. This approach showed that contigs from Amblyomma ticks formed distinct clades (1 and 2). These clades did not present similarities with a specific type of flagelliform silk, a similarity consistently found in the Brevirostrata ticks D andersoni, R. microplus, R. appendiculatus and R. sanguineus. This can be observed in the most distinct clade formed by these latter species of ticks (clade 3). All sequences from this clade present similarity with flagelliform silk. Moreover, sequences in the dendogram displayed a wide-spread dis- tribution, including a clade formed only by sequences from our libraries (clade 4), showing that we have con- tributed with diversified sequences encoding glycine-rich proteins from our ESTs database. Cement-like proteins Contigs homologous to so called cement-like proteins were found among each of the three libraries, yet were most abundant in the library derived from R. microplus (9 contigs versus two in SGFAc and 1 from SGFRs). The cement-like sequences from the same species analyzed before were aligned and used to generate the phyloge- netic tree shown in Figure 5, which showed six distinct clades. Clades 1 and 2 contain cement-like proteins from other Brevirostrata and Longirostrata ticks, indicating the diversity among cement-like proteins. Most sequences from clade 2 presented similarity with the sali- vary gland-associated protein 64P from R. appendicula- tus, except sequence Rm 234, which showed similarity with NPL-2 (neuroprotein-like) from Ixodes pacificus. Clade 1 contains a subclade with sequences derived exclusively from R. microplus. Clade 3, in turn, presented sequences similar to putative cement protein RIM36 from R. appendiculatus (Ac 52) and Unknown protein from R. haemaphysaloides (Rs 156, 17, 4238; Rm 85, 479 and Ac 9). Clades 4 and 5, aside from illustrating the extensive diversity in the expression of cement-like pro- teins between the ticks, also indicate the expansion of the Page 11 of 17 genus Rhipicephalus showing similarity with cement-like antigen protein from Haemaphysalis longicornis and an unknown protein from R. haemaphysaloides. Finally, Clade 6 contains sequences derived exclusively from R. appendiculatus. Examination of the best hits to the sequences in the general NCBI database showed that several GRP contigs were significantly homologus to GRPs of plants (Rm 265, Rm 36, Rm 77, Rs 70, Ac 147, Ac 14) vertebrate skin (Rs 26, Rs12, Rs 70, Ac 354, Ac 13, Ac16) nucleic-acid-bind- ing proteins (Rm 32, Ac 233 ) and to the Mycobacterium tuberculosis PE-PGRS multigene family (contigs Rm 479, Rm 533, Rm 29, Rs 29). These similarities may also shed light on the biological functions of the tick GRPs. In plants many GRPs form the walls of initially polysaccha- ride-rich primary water pipes of elongating plant organs [19]. These functions remit to those of the cement cone in Brevirostrata ticks, which forms a continuation of the hypostome that penetrates the host skin. Interestingly, seed plant GRPs can be allergens for vertebrates [20] and similarly tick saliva can elicit local hypersensitivity reac- tions in immune hosts [21]. GRPs also play a role in regu- lating permeability and penetration of toxins in insect cuticles [9]. In ticks the cement cone may assist the cuti- cle of the hypostome in trapping host cells and molecules that are cytotoxic for the parasite. Many secreted salivary GRPs are similar to RNA-binding proteins, which in the tick may participate in modifying the extracellular traps comprised of nucleic acids that can be produced by mast cells and neutrophils [22], which are present in the local inflammatory infiltrate elicited in the host's skin by tick bites. This finding can also explain the significant quan- tity of transcripts from SGFRm (20.7%, Additional file 1) categorized such as "nucleic acid binding" based on the GO database. Tick GRPs similar to keratins and loricrins, which are major envelope components of terminally dif- ferentiated epithelial cells of vertebrate skin [23], may serve as decoys for the host. Interestingly, the Breviro- strata ticks herein analyzed (R. microplus and R. san- guineus) displayed a greater number of transcripts related to development of epidermis and organization and bio- genesis of extracellular matrix based on homologies to the GO database than the Longirostrata tick (A. cajenn- ense). Finally, the products of the PE-PGRS multigene family of M. tuberculosis form a source of antigenic varia- tion among different strains of this bacterium [24]; in addition PE-PGRS contain many Gly-Ala repeats, which are also present in tick GRPs and which have been shown to inhibit ubiquitin/proteasome-dependent protein deg- radation in mycobacteria and Epstein-Barr virus [25,26]. Since libraries were constructed from a pool of salivary glands from several individual females, the diversity in contigs of salivary GRPs may reflect the existence of a similar mechanism in ticks.