A total of 1440 plaque phages were sequenced from each of the three salivary gland libraries to generate 5' Expressed Sequence Tags (ESTs). A total of approximately 2,900 high quality sequences, including 1,152 from the salivary glands of female R. microplus (SGFRm), 824 from salivary glands of female R. sanguineus (SGFRs) and 929 from salivary glands of female A. cajennense (SGFAc). Redundant sequences were clustered into related groups using BLASTN and then assembled into contiguous sequences yielding 1,406 unique contigs of which 245 were derived from two or more ESTs (transcripts) and 1,165 were derived from a single EST (singleton). As seen in Table , GRPs are abundantly expressed in the salivary glands, ranging from 3- 6% of the total contigs sequenced from these libraries. The SGFRm library contained more ESTs similar to genes coding for GRPs than the other two libraries. The SGFRm and SGFRs libraries exhibit a similar number of ESTs for GRPs (57 and 47 ESTs, respectively), but comparing the number of unique contigs similar to GRPs, SGFRm contained almost double the number (n = 33) of unique contigs as SGFRs (n = 16) and triple that of SGFAc (n = 11). This finding shows that saliva of R. microplus, a Brevirostrata, one-host tick, contains twice as many different GRPs than the other two species of ticks examined herein, one a Brevirostrata, three host tick, the other a Longirostrata, three 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) relative to the other two libraries (57 and 47 ESTs, from SGFRm and SGFRs, respectively).
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).
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 ). 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 protein containing 25% glycine obtained from R. haemaphysaloides 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). Proteins annotated as cement and cement-like proteins from H. longicornis, I. scapularis and R. appendiculatus were also commonly observed among the three libraries (Table ).
Description of matches with glycine-rich proteins present in the Arachnida protein database for transcripts from A. cajennense, R. sanguineus and R. microplus
Comparing the BLAST results of the three libraries shows that, with 33 contigs representing 57 ESTs, R. microplus contained the most abundant contigs homologous to GRPs as compared to 11 contigs from A. cajennenes 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 ).
Differential expression of GRPs in Brevirostrata and Longistrata, and monoxenous and heteroxenous ticks
As reported above, the distribution of GRPs in three Ixodid ticks differed according to the species. In order to better display this distribution, Figure 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 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. sanguineus 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 and Table ). We also analyzed the distribution of 21 types of GRPs among the three libraries. As shown in Figure , 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 numbers in parentheses)]; R. microplus contains twice the amount of contigs encoding GRPs (SGFRm, SGFRs and SGFAc contained 33, 16 and 11 contigs of GRPs, respectively).
Venn diagram showing the distribution of glycine rich protein (GRP) ESTs among three species of hard ticks, A. cajennense, R. sanguineus and R. microplus. Numbers in parentheses represent types of GRP according to the classification in Table 2.
A protein previously described in Rhipicephalus haemaphysaloides haemaphysaloides
(gi 45479213), annotated as of "unknown function", represented the class of GRP with which the majority of ESTs in the 3 libraries presented similarity (Table ). Over half of these transcripts derived from the library from R. microplus
salivary 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-associated protein 64P from Rhipicephalus appendiculatus
ticks (gi 20069012), at least on the first 10 best hits, contrary to what was found for SGFRs and SGFAc. 64P is a GRP of interest because it is a potentially protective antigen for some species of host [7
]. 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
], and salivary homologues are present in several species of ticks [7
As noted above, the annotation of the three cDNA libraries using the BLAST results permitted us to observe that the libraries and some contigs contained fewer or more transcripts and sequences coding for proteins similar to GRPs than expected from a random distribution, as evaluated with the χ2 or Fisher exact tests. Table presents the distribution of all transcripts coding for the GRPs observed among the three salivary gland libraries from females of the tick species studied herein, R. microplus, R. sanguineus and A. cajennense. SGFRm and SGFRs libraries contain significantly (P = 0.006 and P = 0.003, respectively; χ2 test) more transcripts coding for all types of GRPs than the SGFAc library; transcripts for GRPs were equally represented in the SGFRm and SGFRs libraries (P = 0.821, χ2 test). These results show that for Brevirostrata ticks, R. microplus and R. sanguineus, the expression in salivary glands of all types of GRPs was significantly higher than in those of a Longirostrata tick (A. cajennense). The R. microplus salivary gland library contained more types and transcripts of proteins similar to GRPs than the SGFRs, but the difference in the proportions of the GRPs did not quite reach significance with the present depth of sequencing (P = 0.072, χ2 test; Table ). 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. cajennense (Table ). 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 mechanisms. 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.
Differential Abundance of Transcripts and Diversity of Types of GRPs in Salivary Gland Libraries from females of Rhipicephalus (Boophilus) microplus SGFRm), Rhipicephalus sanguineus (SGFRs)and Amblyomma cajennense (SGFAc)
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 abundant 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 ). A contig coding for a GRP similar to "acanthoscurrin 1 precursor" from Acanthoscurria gomesiana spiders (Genbank accession gi 27524417) was also not significantly differentially represented (Table ), although SGFRs contains 6 ESTs and SGFAc has just 1 EST. However, the GRP similar to "salivary gland-associated protein 64P" from R. appendiculatus (a Brevirostrata, heteroxenous tick), regarded as a cement protein, was significantly 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 transcripts that were similar to silks of true spiders (Araneomorphae; Table ), 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 preformed 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 expression 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. Furthermore, 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 salivation.
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% of the terms and SGFAc with 26.1% versus 12.1% of terms for SGFRs). Glycine-rich proteins related to epidermal development have also been found in others arthropods, such as the silkworm Bombix mori
]. Interestingly terms related to development of epidermis were the most abundant category of all, assigned to SGFRs (70.2%), a library made from a heteroxenous, Brevirostrata tick. Over half (52.2%) of the terms assigned for SGFAc fell into the category "unknown", reflecting the fact that little information is available about biological characteristics of saliva from A. cajennense