Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 Page 6 of 17 Figure 1 Venn diagram showing the distribution of glycine rich protein (GRP) ESTs among three species of hard ticks, A. cajenn- ense, R. sanguineus and R. microplus. Numbers in parentheses repre- sent types of GRP according to the classification in Table 2. expression in salivary glands of all types of GRPs was sig- nificantly higher than in those of a Longirostrata tick (A. cajennense). The R. microplus salivary gland library con- tained more types and transcripts of proteins similar to GRPs than the SGFRs, but the difference in the propor- tions of the GRPs did not quite reach significance with the present depth of sequencing (P = 0.072, χ2 test; Table 3). Nevertheless, these results show that a Brevirostrata, monoxenous tick, which remains attached to the same host for at least three weeks, relies on a greater variety of GRPs than the Brevirostrata heteroxenous tick examined in this study. On the other hand, the library from R. microplus contained significantly (P = 0.015, χ2 test) more transcripts of GRPs than the library from the salivary glands of the Longirostrata, heteroxenous tick, A. cajenn- ense (Table 3). This finding suggests that in order to feed on a single host for up to three weeks, monoxenous ticks with short mouthparts must be equipped to deal with a larger repertoire of the host's local homeostatic mecha- nisms. It is noteworthy that by the time the monoxenous tick R. microplus completes its blood meal its host will have mounted an adaptive immune response. The greater diversity of GRPs in this species may reflect a form of antigenic variation. We also observed that the distribution of ESTs within some contigs was greater in a given species of tick. Contig 29 from SGFRm, coding for a protein similar to an "unknown" protein from R. h. haemaphysaloides ticks (Genbank accession: gi 45479213), was the most abun- dant transcript among the three libraries and the most abundant in the SGFRm library, with 21 ESTs versus 10 ESTs in both the SGFRs and SGFAc libraries, however it was not differentially represented among the three ticks (Table 3). A contig coding for a GRP similar to "acan- thoscurrin 1 precursor" from Acanthoscurria gomesiana spiders (Genbank accession gi 27524417) was also not significantly differentially represented (Table 3), although SGFRs contains 6 ESTs and SGFAc has just 1 EST. How- ever, the GRP similar to "salivary gland-associated pro- tein 64P" from R. appendiculatus (a Brevirostrata, heteroxenous tick), regarded as a cement protein, was sig- nificantly more expressed in salivary glands of female R. sanguineus than in those of A. cajennense (P = 0.001, χ2 test). This result suggests that females of a Brevirostrata, heteroxenous tick rely more on this protein to attach and feed on their last host than Longirostrata, heteroxenous ticks. Finally, regarding the nature of similarities, it was interesting to note that R. microplus, R. sanguineus and A. cajennense expressed, respectively, 23, 17 and 8 tran- scripts that were similar to silks of true spiders (Araneo- morphae; Table 2), however the differences in distribution did not reach statistical significance. Our results do not preclude the fact that some of the GRPs for which transcripts were not detected in a given species may indeed be present in salivary glands as pre- formed proteins stored in granules. Nevertheless, this still represents a biological difference involving GRPs that is reflected in the transcription profile. On the other hand, previous work [12] clearly shows that tick salivary glands are not completely "pre-loaded" and ready to secrete when a tick attaches to a host. Indeed, the expres- sion of at least 27 genes encoding secreted proteins increases in salivary glands of female Ixodes scapularis ticks after attachment to their host and, interestingly, almost a third (eight) of these encode GRPs. Further- more, transcripts for GRPs were not observed in salivary glands from unfed females. Kaufman [13] showed that fluid secretion by salivary glands was similar in the females of several species of Ixodidae ticks, including Brevirostrata and Longirostratata ticks suggesting that salivation is similar throughout the Ixodid family [13], i.e., if the presence of 'pre-loaded' granules has a determinant role in salivation, that work would have found differences for distinct tick species, mainly at the early phases of sali- vation. Besides analyses performed with the NCBI database, we used the gene ontology (GO) database to categorize the GRP contigs from individual libraries. Results must be interpreted with caution since the GRP sequences are of low complexity and GO categories are still not entirely comprehensive for all biological functions. Nevertheless, differences were seen among the three species of ticks. The GRP transcripts were categorized into GO terms for nine biological processes (Additional file 1); the term "epidermis development" was most frequently assigned to transcripts from heteroxenous ticks (SGFRs with 70.2%