Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 Page 3 of 17 Table 1: Characteristics cDNA libraries constructed using salivary glands (SG) dissected from three feeding female Ixodid ticks: Rhipicephalus sanguineus (Rs), Rhipicephalus (Boophilus) microplus (Rm) and Amblyomma cajennense (Ac). Library SGFRm SGFRs SGFAc a Percent of total ESTs 1,152 874 929 Contigs 533 455 418 ESTs/ Transcripts 2.16 1.92 2.22 ESTs encoding GRPs 57 (4.94)a 47 (5.37)a 23 (2.90)a Contigs of GRPs 33 (6.19)a 16 (3.51)a 11 (3.82)a Average n° GRP ESTs/ Contig of GRP 1.72 2.93 2.09 host tick. The SGFAc library contained approximately the same proportion of unique GRP contigs as the SGFRs library, however these are formed by fewer ESTs (23) rela- tive to the other two libraries (57 and 47 ESTs, from SGFRm and SGFRs, respectively). Comparison of library-derived glycine rich proteins to published and custom databases Comparsion of the contigs from the three libraries with a customized database of all Arachnida proteins found in Genbank revealed that 60 contigs had similarities with 21 different types of GRP, based on published annotated sequences (Table 2). Contigs were considered to encode GRPs if the translated amino acid sequence contained a glycine content of at least 20% (with three exceptions among the 60 unique contigs, which contained 11 and 17% glycine). The most abundant GRP (41 total ESTs) found among all three libraries was a 506 amino acid pro- tein containing 25% glycine obtained from R. haemaphys- aloides and annotated as "unknown function". Flagelliform silk proteins (~50% glycine), identified from various spider species, was the second most abundant GRP found among the three libraries (n = 23 ESTs). Pro- teins annotated as cement and cement-like proteins from H. longicornis, I. scapularis and R. appendiculatus were also commonly observed among the three libraries (Table 2). Comparing the BLAST results of the three libraries shows that, with 33 contigs representing 57 ESTs, R. microplus contained the most abundant contigs homolo- gous to GRPs as compared to 11 contigs from A. cajen- nenes and 16 contigs from R. sanguineus. Salivary glands from R. microplus also contained the greatest variety of GRP with contigs homologous to 11 different GRPs whereas A. cajennense and R. sanguineus salivary glands contained 9 and 8 different GRPs, respectively (Table 2). Differential expression of GRPs in Brevirostrata and Longistrata, and monoxenous and heteroxenous ticks As reported above, the distribution of GRPs in three Ixo- did ticks differed according to the species. In order to better display this distribution, Figure 1 shows a Venn diagram of the numbers of GRP ESTs and types of GRPs (numbers in parentheses) found in common among the three species of ticks studied herein. Figure 1 also shows the number of GRP ESTs and types of GRPs that are unique to each species. More GRP ESTs are expressed uniquely in the salivary glands of females of R. microplus (14 versus 3 and 2 ESTs for, respectively A. cajannense and R. sanguineus). These transcripts represented 8 unique types of GRPs in R. microplus, whereas R. san- guineus and A. cajannense each presented only unique 1 type of GRP. On the other hand, only two GRPs (from a total of 21 types) were common to all three species of ticks and were represented by 50 ESTs (Figure 1 and Table 2). We also analyzed the distribution of 21 types of GRPs among the three libraries. As shown in Figure 1, R. microplus contained almost twice as many types of GRPs than R. sanguineus or A. cajennense [SGFRm: 16 types of GRPs; SGFRs and SGFAc: 9 types of GRPs each (see num- bers in parentheses)]; R. microplus contains twice the amount of contigs encoding GRPs (SGFRm, SGFRs and SGFAc contained 33, 16 and 11 contigs of GRPs, respec- tively). A protein previously described in Rhipicephalus hae- maphysaloides haemaphysaloides (gi 45479213), anno- tated as of "unknown function", represented the class of GRP with which the majority of ESTs in the 3 libraries presented similarity (Table 2). Over half of these tran- scripts derived from the library from R. microplus sali- vary glands (21 from SGFRm, 10 from SGFRs and 10 from SGFAc). Interestingly, the SGFRm library does not present any EST with similarity to salivary gland-associ- ated protein 64P from Rhipicephalus appendiculatus ticks (gi 20069012), at least on the first 10 best hits, con- trary to what was found for SGFRs and SGFAc. 64P is a GRP of interest because it is a potentially protective anti- gen for some species of host [7,10]. The amino acid sequence of the 64P secreted salivary protein is similar to epidermal/dermal keratin and collagen proteins, which are mammalian structural proteins of the skin [8,11], and salivary homologues are present in several species of ticks [7].