Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 Background In order to acquire a blood meal, Ixodid (hard) ticks secrete diverse salivary proteins that inhibit their hosts' defense mechanisms and permit hematophagy to pro- ceed for many days [1]. But ticks must first attach to the skin of their hosts and attachment must be effective for the duration of the tick's blood meal. Ixodid ticks are clas- sified by the number of different hosts they feed on dur- ing the parasitic phase of their life cycle; one host, two hosts or three hosts. Ticks that complete the entire para- sitic cycle on one host are monoxenous parasites, whereas tick that feed on two or more different hosts with an interval off the host between the feeds are het- eroxenous. Success of attachment on one or more hosts depends, among other factors, on the salivary proteins that are believed to form cement cones. These structures fix tick mouthparts to the host's skin and possibly dis- guise and/or lubricate them [2]. The architecture of the cement cone depends on the both the depth of penetra- tion of the tick's hypostome into the host's skin and the degree to which cement encases the hypostome. The cat- tle tick, Rhipicepahalus microplus, and the brown dog tick, R. sanguineus, are classified as Brevirostrata ticks because their mouthparts are short and barely penetrate into the epidermis of theirs hosts. These parts are there- fore assisted by a wide cement cone that reaches more deeply into this layer of skin and also extrudes the epider- mis [3]. Consequently, the cement cone of Brevirostrata ticks tends to be wide and deep, completely surrounding the hypostome and extruding above the epidermis of the host skin [4]. Histological cross-sections of an adult R. sanguineus attached to a dog clearly illustrate the superfi- cial penetration of the hyposotome and the extensive cement cone which appears to "glue" the mouthparts in place [5]. R. microplus is a monoxenous tick and its life cycle, spent on a single host, is of approximately 21 days; R. sanguineus is a heteroxenous tick. Conversely, Ambly- omma cajennense, also a heteroxenous parasite, is a Lon- girostrata tick with its long hypostome fully penetrating well into its host's dermis and encased by a narrow cement cone [3]. Several salivary proteins present in the tick's cement cone are rich in glycine (glycine-rich pro- teins, GRPs) [3,6,7]. GRPs are abundant in nature and constitute a large family of heterogeneous proteins enriched in glycine residues by various proportions, occupying from 20% to 70% of the total amino acid resi- dues of the protein. GRPs can be classified into several groups based on their molecular structure [8,9]. During the course of our studies of the transcriptome of salivary glands from R. microplus, R. sanguineus and A. cajennense we annotated different types of GRPs and observed that these contigs represent from 3 to over 6% of the total number, higher than any other class of pro- tein. Furthermore, we observed that the distribution and Page 2 of 17 abundance of the contigs and the number of transcripts that form them differed according to the species. Since proteins isolated from the cement cone are rich in glycine and this structure may have a role in attachment and since the various species of ticks have different require- ments for attachment, herein we perform initial tests of the hypothesis that there are not only anatomical, but also chemical differences between the cement cones pro- duced by these three species of ticks. These differences could vary according to their biology, such as whether they infest one or more hosts and whether anatomy of their mouthparts comprises short or long hypostomes. We constructed three non-normalized, PCR-based cDNA libraries from the salivary glands of female R. san- guineus, R. microplus and A. cajennense and analyzed the expressed sequence tags (ESTs) obtained using custom- ized bioinformatics software. We observed that the expression of contigs and their transcripts coding for gly- cine-rich proteins differed in quantity as well in diversity, depending on the species of the tick. In order to further test this hypothesis we also performed a phylogenetic analysis using the sequences from our work as well as of publicly available sequences from all the available sia- lomes of other species of heteroxenous and Longirostrata or Brevistrata ticks that have been annotated as GRPs. Results and Discussion Library Construction 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 approxi- mately 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 (tran- scripts) and 1,165 were derived from a single EST (single- ton). As seen in Table 1, GRPs are abundantly expressed in the salivary glands, ranging from 3- 6% of the total con- tigs 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 Brevi- rostrata, three host tick, the other a Longirostrata, three