BACKGROUND
in order to acquire a blood meal, ixodid  ticks secrete diverse salivary proteins that inhibit their hosts' defense mechanisms and permit hematophagy to proceed for many days  <cit> .
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 classified by the number of different hosts they feed on during the parasitic phase of their life cycle; one host, two hosts or three hosts.
ticks that complete the entire parasitic 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 heteroxenous.
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 disguise and/or lubricate them  <cit> .
the architecture of the cement cone depends on the both the depth of penetration of the tick's hypostome into the host's skin and the degree to which cement encases the hypostome.
the cattle 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 therefore assisted by a wide cement cone that reaches more deeply into this layer of skin and also extrudes the epidermis  <cit> .
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  <cit> .
histological cross-sections of an adult r. sanguineus attached to a dog clearly illustrate the superficial penetration of the hyposotome and the extensive cement cone which appears to "glue" the mouthparts in place  <cit> .
r. microplus is a monoxenous tick and its life cycle, spent on a single host, is of approximately  <dig> days; r. sanguineus is a heteroxenous tick.
conversely, amblyomma cajennense, also a heteroxenous parasite, is a longirostrata tick with its long hypostome fully penetrating well into its host's dermis and encased by a narrow cement cone  <cit> .
several salivary proteins present in the tick's cement cone are rich in glycine   <cit> .
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 residues of the protein.
grps can be classified into several groups based on their molecular structure  <cit> .
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  <dig> to over 6% of the total number, higher than any other class of protein.
furthermore, we observed that the distribution and 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 requirements for attachment, herein we perform initial tests of the hypothesis that there are not only anatomical, but also chemical differences between the cement cones produced 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. sanguineus, r. microplus and a. cajennense and analyzed the expressed sequence tags  obtained using customized bioinformatics software.
we observed that the expression of contigs and their transcripts coding for glycine-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 sialomes of other species of heteroxenous and longirostrata or brevistrata ticks that have been annotated as grps.
RESULTS
library construction
a total of  <dig> plaque phages were sequenced from each of the three salivary gland libraries to generate 5' expressed sequence tags .
a total of approximately  <dig>  high quality sequences, including  <dig>  from the salivary glands of female r. microplus ,  <dig> from salivary glands of female r. sanguineus  and  <dig> from salivary glands of female a. cajennense .
redundant sequences were clustered into related groups using blastn and then assembled into contiguous sequences yielding  <dig>  unique contigs of which  <dig> were derived from two or more ests  and  <dig>  were derived from a single est .
as seen in table  <dig>  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 , but comparing the number of unique contigs similar to grps, sgfrm contained almost double the number  of unique contigs as sgfrs  and triple that of sgfac .
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  relative to the other two libraries .
a percent of total
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  <dig> contigs had similarities with  <dig> different types of grp, based on published annotated sequences .
contigs were considered to encode grps if the translated amino acid sequence contained a glycine content of at least 20% .
the most abundant grp  found among all three libraries was a  <dig> amino acid protein containing 25% glycine obtained from r. haemaphysaloides and annotated as "unknown function".
flagelliform silk proteins , identified from various spider species, was the second most abundant grp found among the three libraries .
proteins annotated as cement and cement-like proteins from h. longicornis, i. scapularis and r. appendiculatus were also commonly observed among the three libraries .
* computation of glycine content was calculated based on translated consensus sequence
comparing the blast results of the three libraries shows that, with  <dig> contigs representing  <dig> ests, r. microplus contained the most abundant contigs homologous to grps as compared to  <dig> contigs from a. cajennenes and  <dig> contigs from r. sanguineus.
salivary glands from r. microplus also contained the greatest variety of grp with contigs homologous to  <dig> different grps whereas a. cajennense and r. sanguineus salivary glands contained  <dig> and  <dig> different grps, respectively .
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  <dig> shows a venn diagram of the numbers of grp ests and types of grps  found in common among the three species of ticks studied herein.
figure  <dig> 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 .
these transcripts represented  <dig> unique types of grps in r. microplus, whereas r. sanguineus and a. cajannense each presented only unique  <dig> type of grp.
on the other hand, only two grps  were common to all three species of ticks and were represented by  <dig> ests .
we also analyzed the distribution of  <dig> types of grps among the three libraries.
as shown in figure  <dig>  r. microplus contained almost twice as many types of grps than r. sanguineus or a. cajennense ; r. microplus contains twice the amount of contigs encoding grps .
a protein previously described in rhipicephalus haemaphysaloides haemaphysaloides , annotated as of "unknown function", represented the class of grp with which the majority of ests in the  <dig> libraries presented similarity .
over half of these transcripts derived from the library from r. microplus salivary glands .
interestingly, the sgfrm library does not present any est with similarity to salivary gland-associated protein 64p from rhipicephalus appendiculatus ticks , at least on the first  <dig> 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  <cit> .
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  <cit> , and salivary homologues are present in several species of ticks  <cit> .
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 χ <dig> or fisher exact tests.
table  <dig> 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  more transcripts coding for all types of grps than the sgfac library; transcripts for grps were equally represented in the sgfrm and sgfrs libraries .
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 .
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 .
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  more transcripts of grps than the library from the salivary glands of the longirostrata, heteroxenous tick, a. cajennense .
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.
*chi-square test for representation of the glycine-rich proteins distributed in the three cdna libraries.
we also observed that the distribution of ests within some contigs was greater in a given species of tick.
contig  <dig> from sgfrm, coding for a protein similar to an "unknown" protein from r. h. haemaphysaloides ticks , was the most abundant transcript among the three libraries and the most abundant in the sgfrm library, with  <dig> ests versus  <dig> ests in both the sgfrs and sgfac libraries, however it was not differentially represented among the three ticks .
a contig coding for a grp similar to "acanthoscurrin  <dig> precursor" from acanthoscurria gomesiana spiders  was also not significantly differentially represented , although sgfrs contains  <dig> ests and sgfac has just  <dig> est.
however, the grp similar to "salivary gland-associated protein 64p" from r. appendiculatus , regarded as a cement protein, was significantly more expressed in salivary glands of female r. sanguineus than in those of a. cajennense .
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,  <dig>   <dig> and  <dig> transcripts that were similar to silks of true spiders , 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  <cit>  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  <dig> genes encoding secreted proteins increases in salivary glands of female ixodes scapularis ticks after attachment to their host and, interestingly, almost a third  of these encode grps.
furthermore, transcripts for grps were not observed in salivary glands from unfed females.
kaufman  <cit>  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  <cit> , 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  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 ; the term "epidermis development" was most frequently assigned to transcripts from heteroxenous ticks .
glycine-rich proteins related to epidermal development have also been found in others arthropods, such as the silkworm bombix mori  <cit> .
interestingly terms related to development of epidermis were the most abundant category of all, assigned to sgfrs , a library made from a heteroxenous, brevirostrata tick.
over half  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 ticks.
phylogenetic analyses of glycine-rich proteins
silk-like proteins
phylogenetic analysis of grp contigs from the three ticks studied herein shows  <dig> distinct clades  as displayed in figure  <dig>  two of them contain a group of contigs that presented similarities with silk proteins of spiders.
one was similar to a flagelliform silk protein, , which is a viscous, glue-like silk from orb-weaving spiders of the nephila genus; another was similar to major ampullate spidroin  from kukulcania hibernalis, a cribellate , non-orb-weaving spider.
interestingly, these clades are the most defined in the dendogram, as may be observed through the proximity among members of these clades.
another feature of clades  <dig> and  <dig> is that each is formed by contigs from a single species of tick: clade  <dig> presented contigs only from a. cajennense and clade  <dig> contained contigs only from r. microplus, probably for this reason the clades showed better proximity among the members.
this finding is compatible with our hypothesis that metastriate ticks presenting with different biological characteristics rely on different types of grps.
noteworthy is the fact fsp is an elastic and glue -like adhesive silk  <cit> , a characteristic which could be important for a brevirostrata monoxenous tick.
the third distinct clade has showed similarity with an unknown protein  from r. haemaphysaloides and contains contigs from only brevirostrata ticks, i.e., r. microplus and r. sanguineus, again a finding that is compatible with our hypothesis.
futhermore, although distinct clades were not formed by the remaining grp contigs, we noted that contigs segregated into two different patterns: those which presented matches with spider silk proteins  are concentrated in clades that are distant from those that have similarities with cement-like proteins of ticks , with the exception of contigs rs <dig>  rm <dig> and ac <dig>  this finding suggests that these two "major types" of grps  may have different roles during attachment ticks on the host.
the similarities found with spider grps were also with masps and fsps, albeit from different genera of spiders.
interestingly, a. cajennense was represented only twice among the cement-like  contigs, while two other cement-like contigs from this species grouped with spider silk-like contigs.
a multiple alignment analyses of the three distinct clades  showed the level of conservation among contigs, as can be observed in figure  <dig>  clade  <dig>  formed by contigs from sgfrm showed the greatest degree of conservation.
sequence alignment of rm <dig> and rm <dig> show an identity of  <dig> % and  <dig> % similarity.
rm <dig> and rm <dig> presented the lowest conservation, 33% identity and  <dig> % similarity.
contigs of clade  <dig> formed by sgfac sequences also displayed low divergence, even the least similar transcript ac14; ac <dig> presented a reasonable equivalence between sequences  whereas ac <dig> and ac <dig> had better conservation, presenting an identity of  <dig> % and similarity of  <dig> %.
clade  <dig> did not did not present the same level of conservation as clades  <dig> and  <dig>  except for contigs rs  <dig> and rm <dig>  which presented a high identity and similarity of  <dig> % and  <dig> %, respectively.
it was interesting to note the large conserved region visualized in each of the sequence alignments of clades  <dig> and  <dig>  five conserved regions are encountered in sgfrm contigs of clade  <dig> that are identical in the  <dig> contigs: 1) qlgps , 2) sgslg , 3) gvlpsg , 4) sgvgrg  and 5) tgfvlpg ; some of these conserved regions could be extend if the charge and chemical proprieties of the residues from different contigs are taken into consideration.
in the alignment for clade  <dig>  aspartic acid  could change to glutamic acid  at residue  <dig>  leucine  to isoleucin  at residue  <dig> , glycine  to serine  at residue  <dig>  and valine  to alanine  at residue  <dig>  and these amino acids present similar characteristics among each other.
the same aspects can be observed for residues 43- <dig> and 89- <dig>  in addition, when the contigs of clade  <dig> are compared with the composition of flageliform silk proteins, positions of important residues of silk proteins such as glycine, serine and proline are conserved among them .
regarding clade  <dig>  two conserved regions are found in all sgfac contigs, one composed of  <dig> residues, fgsgf , and a second one with  <dig> residues, sglgggygsg .
it is noteworthy that both regions have glycine  and serine residues.
again, conserved regions contain glycine and serine residues, two abundant amino acids in spider silk.
alignment of ac contigs and masp proteins showed similarity in most positions containing glycine and serine residues, but not in positions with proline residues .
sheets are formed in secondary structures of silk proteins with repeats containing glycine, serine and alanine, which confer their elastic and strength proprieties  <cit> .
the presence of a proline residue between serine and glycine, as happens in sequences of clade  <dig>  could be important to "interrupt" secondary structures determined by glycine, serine and alanine, promoting acquisition of more elastic and less stiff properties.
the mechanical property of elasticity is greater in flagelliform silk proteins of orb-weaving spiders  that are made to capture flying prey than in major ampullate spidroin silk proteins  used in capture threads in less mobile spiders  <cit> .
the multiple alignments of clade  <dig> sequences, which are homologous with an "unknown" protein from rhipicephalus haemaphysaloides did not present conserved regions, perhaps owing to divergence among contigs and many gaps that could not allow long conserved regions.
however, it can be observed though shading of the alignments that they present similarities as described before, with regions abundant in glycine and serine.
in addition to the contigs derived from r. microplus, r. sanquineus and a. cajennense analyzed herein and in order to increase the stringency of the test for our hypothesis, we performed a multiple alignment using contigs from the work of francischetti et al.
<cit>  that reviewed all of the available salivary components of ticks.
this work described a superfamily of glycine-rich proteins for argasid and ixodid ticks .
we observed in this work that argasid ticks produce only three types out of over four hundred types of grps.
this maybe due to the fact that argasid ticks are rapid feeders and complete a blood meal in minutes.
we also observed that the majority of the grps found in prostriate ticks , are collagen-like proteins.
this group appears to have a primitive form of attachment among the ixodid ticks  <cit> , presenting an intermediate complexity in this process.
finally, this work showed that in metastriate ticks  the grps belong to ggy, gyg and metastriate spider-like cement protein families.
we therefore excluded analyses of grps from ixodes sp.
and argasidae ticks and selected grps from the nr database on ncbi that present similarities with silk-like and cement-like proteins from a. variegatum, a. americanum, d. andersoni, r. microplus and r. appendiculatus .
we aligned all sequences similar to silk-like proteins from our libraries and the nr database from sup-table  <dig>  and using the neighbor joining analysis produced the phylogram shown in figure  <dig>  sequences generated by the present work are symbolized with a circle , a square  and a triangule .
in addition to our sequences,  <dig> other transcript sequences of other species ticks are represented: dermacentor andersoni and r. appendiculatus, both brevirostrata, heteroxenous 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 representatives of longirostrata, monoxenous ticks that could be included in this analysis.
this approach showed that contigs from amblyomma ticks formed distinct clades .
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 .
all sequences from this clade present similarity with flagelliform silk.
moreover, sequences in the dendogram displayed a wide-spread distribution, including a clade formed only by sequences from our libraries , showing that we have contributed 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 .
the cement-like sequences from the same species analyzed before were aligned and used to generate the phylogenetic tree shown in figure  <dig>  which showed six distinct clades.
clades  <dig> and  <dig> contain cement-like proteins from other brevirostrata and longirostrata ticks, indicating the diversity among cement-like proteins.
most sequences from clade  <dig> presented similarity with the salivary gland-associated protein 64p from r. appendiculatus, except sequence rm  <dig>  which showed similarity with npl- <dig>  from ixodes pacificus.
clade  <dig> contains a subclade with sequences derived exclusively from r. microplus.
clade  <dig>  in turn, presented sequences similar to putative cement protein rim <dig> from r. appendiculatus  and unknown protein from r. haemaphysaloides .
clades  <dig> and  <dig>  aside from illustrating the extensive diversity in the expression of cement-like proteins between the ticks, also indicate the expansion of the genus rhipicephalus showing similarity with cement-like antigen protein from haemaphysalis longicornis and an unknown protein from r. haemaphysaloides.
finally, clade  <dig> 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  vertebrate skin  nucleic-acid-binding proteins  and to the mycobacterium tuberculosis pe-pgrs multigene family .
these similarities may also shed light on the biological functions of the tick grps.
in plants many grps form the walls of initially polysaccharide-rich primary water pipes of elongating plant organs  <cit> .
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  <cit>  and similarly tick saliva can elicit local hypersensitivity reactions in immune hosts  <cit> .
grps also play a role in regulating permeability and penetration of toxins in insect cuticles  <cit> .
in ticks the cement cone may assist the cuticle 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  <cit> , which are present in the local inflammatory infiltrate elicited in the host's skin by tick bites.
this finding can also explain the significant quantity of transcripts from sgfrm  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 differentiated epithelial cells of vertebrate skin  <cit> , may serve as decoys for the host.
interestingly, the brevirostrata ticks herein analyzed  displayed a greater number of transcripts related to development of epidermis and organization and biogenesis of extracellular matrix based on homologies to the go database than the longirostrata tick .
finally, the products of the pe-pgrs multigene family of m. tuberculosis form a source of antigenic variation among different strains of this bacterium  <cit> ; 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 degradation in mycobacteria and epstein-barr virus  <cit> .
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.
grps present biochemical characteristics that could possibly be involved in stabilizing the tick to its feeding site for long periods due to their putative structural and mechanical functions inferred from the abundance of the amino acid glycine.
grps may also block host immune system molecules that enter in contact with the tick mouthparts  <cit> .
many contigs were similar to silk proteins from spiders, such as fibroin, dragline, flagelliform, major ampullate spidroin and flag silks.
each one of these fibers is composed of one or more proteins encoded by the silk fibroin gene family.
spiders draw fibers from dissolved fibroin proteins that are stored in specialized sets of abdominal glands  <cit> .
it is interesting to note that ticks generate silk-like proteins from their salivary glands, while spiders use abdominal glands for this purpose and reserve their salivary glands for production of venom.
tick silk-like grps may possibly support mechanical needs , as well as the capture of prey and predators .
spider silks are being employed as scaffolds for engineering tissues  <cit>  and tick silk-like proteins may be more adequate for this purpose because of the intimate relation of this parasite with its host's skin.
there are other precedents in nature for our finding that the distribution of distinct grps correlates with the biology of metastriate ticks.
spiders, which are also arachnidae, offer a well known example: the architecture and mechanical properties of different spider webs are correlated with the biological characteristics of their spinners, for example, aerial versus terrestrial capture habits.
these properties ultimately rely on the specialized functions of different types of silks.
of interest to studies on the evolution of ticks, orb weaving by spiders is monophyletic, having evolved only once and speciation of spiders relates to use of different silks  <cit> .
genes encoding flagelliform silks were thought to be expressed exclusively by modern orb weaver spiders that make more elastic, gluey webs.
however it was recently shown that cribellate orb weavers, which make drier webs, also express flagelliform silk genes  <cit> , albeit in lower quantities.
blackledge and colleagues  <cit>  suggested that an increase in the expression of flagelliform silk genes may have resulted in development of modern orb weavers  <cit> .
another example refers to the silks produced by salivary glands of simuliid filter-feeding flies.
simulium noelleri and s. ornatum use silk pads to attach to substrates, the composition of which varies according the requirements of their habitats: s. noelleri feeds in lake outlets where weaker currents are found and s. ornatum feeds in open waters with stronger currents.
accordingly, there are differences between ageing processes and biochemical composition of the silk pads from these two species, s. ornatum presenting the most durable structure  <cit> .
a third and final example is offered by larvae of two species of caddisflies.
hydropsyche angustipennis spins hiding tubes and catching nets that collect food in water currents; larvae of limnephilus decipiens use silk fiber only for stitching fragments of grass into hiding and pupation cases.
the composition of the silk fibers from these species differed by the arrangement of motifs in higher order repeats and by the presence of species-specific motifs.
although the amounts of glycine are similar, the h-fibroin of h. angustipennis presents proline containing motifs, whereas l. decipiens presents a highly charged motif, eegrrr  <cit> .
CONCLUSIONS
in the present work the differences observed for distribution of glycine-rich proteins were related to the number of hosts visited  and to the anatomy of mouthparts  of three species of metastriate ticks.
all ixodid ticks, with the exception of some prostriate, present a strategy for attachment, but it differs among them.
the species from the genus amblyomma, which belongs to the longirostrata ticks, secrete a casing around their long, fully inserted hypostome.
in ticks from the brevirostrata group, which includes species from the genus rhipicephalus, the mouthparts are short and barely penetrate in epidermis  <cit> , so a larger cement cone, from which grps have been purified  <cit> , is necessary and is deposited in the upper layers of their host's skin.
thus, it seems that the two brevirostrata ticks, r. microplus and r. sanguineus, need to express more glycine-rich proteins than the longirostrata tick, a. cajennense, in order to compensate for the small size of mouthparts and for the superficial fixation at the site of attachment.
furthermore, r. microplus is monoxenous and r. sanguineus is heteroxenous and comparisons made between these ticks show that the former presents the greatest diversity of glycine-rich proteins, possibly because it is a one-host tick that feeds uninterrupted for many days until completion of its life cycle and, therefore, has greater demands for sustaining its attachment on host skin.
contigs of salivary glands for several other species of ticks have also been examined.
while the relative abundance of transcripts coding for glycine-rich proteins cannot be accurately compared between salivary gland libraries constructed in different laboratories and undergoing different biological situations , it is still noteworthy that annotation of the transcriptomes of salivary glands from female i. scapularis and i. pacificus indicate that prostriate ticks do not rely on glycine-rich proteins as heavily as metastriate ticks for their attachment to hosts or for other biological functions  <cit> .
on the other hand, salivary glands of females of d. andersoni, a metastriate, heteroxenous, brevirostrata tick, also contain abundant transcripts for grps: of the  <dig> contigs containing the most abundantly expressed ests in salivary glands of females of d. andersoni,  <dig> presented similarities to glycine-rich proteins and contained from  <dig> to  <dig> ests each  <cit> .
in conclusion, our findings furnish preliminary evidence to support the hypothesis that species of ticks with differences in the anatomy of their mouthparts and in the number of hosts they infest during their biological cycle rely on different types and quantities of glycine-rich proteins.
this hypothesis must be further tested by expanding these observations to a larger number of species, by experimental approaches such as rna interference of expression of selected grps and by characterization of isolated grps.
the data suggests that prostriate ticks rely on their elongated barbed hypostome mouthparts and make shallow cement cones, while the metastriate ticks rely on a larger and deeper cement cone possibly to compensate their relatively smaller mouth parts  <cit> .
the number of hosts visited by ticks during the parasitic stage of their life cycle also requires adaptations.
according to balashov   <cit>  and hoogstraal and kim   <cit>  there was a transition from the three host to the two and one host cycle in hyalomma and in rhipicephalinae species of ticks.
the biological characteristic of having a single host is regarded as an adaptation of this immobile ectoparasite to large nomadic animals since ixodid ticks die when they are unable to find a host.
monoxenous ticks are thus better adapted to open environments inhabited by large, grazing ungulates.
the ability to molt on the vertebrate reduces the number of necessary encounters and thus increases chances for tick survival.
in addition to elucidating the biology of tick salivary proteins, the information contained in this work is relevant for the development of vaccines that target grps of ticks and that aim for protection against a broad range of species.
the approach undertaken in this work can subsidize the choice of the different grps present in tick salivary glands for evaluation as protective antigens.

 in order to acquire a blood meal, ixodid ticks secrete diverse salivary proteins that inhibit their hosts' defense mechanisms and permit hematophagy to proceed for many days. 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 classified by the number of different hosts they feed on during the parasitic phase of their life cycle; one host, two hosts or three hosts. ticks that complete the entire parasitic 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 heteroxenous. 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 disguise and/or lubricate them. the architecture of the cement cone depends on the both the depth of penetration of the tick's hypostome into the host's skin and the degree to which cement encases the hypostome. the cattle 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 therefore assisted by a wide cement cone that reaches more deeply into this layer of skin and also extrudes the epidermis. 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. histological cross-sections of an adult r. sanguineus attached to a dog clearly illustrate the superficial penetration of the hyposotome and the extensive cement cone which appears to "glue" the mouthparts in place. r. microplus is a monoxenous tick and its life cycle, spent on a single host, is of approximately days; r. sanguineus is a heteroxenous tick. conversely, amblyomma cajennense, also a heteroxenous parasite, is a longirostrata tick with its long hypostome fully penetrating well into its host's dermis and encased by a narrow cement cone. several salivary proteins present in the tick's cement cone are rich in glycine. 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 residues of the protein. grps can be classified into several groups based on their molecular structure. 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 to over 6% of the total number, higher than any other class of protein. furthermore, we observed that the distribution and 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 requirements for attachment, herein we perform initial tests of the hypothesis that there are not only anatomical, but also chemical differences between the cement cones produced 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. sanguineus, r. microplus and a. cajennense and analyzed the expressed sequence tags obtained using customized bioinformatics software. we observed that the expression of contigs and their transcripts coding for glycine-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 sialomes of other species of heteroxenous and longirostrata or brevistrata ticks that have been annotated as grps.  library construction a total of plaque phages were sequenced from each of the three salivary gland libraries to generate 5' expressed sequence tags. a total of approximately high quality sequences, including from the salivary glands of female r. microplus, from salivary glands of female r. sanguineus and from salivary glands of female a. cajennense. redundant sequences were clustered into related groups using blastn and then assembled into contiguous sequences yielding unique contigs of which were derived from two or more ests and were derived from a single est. 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, but comparing the number of unique contigs similar to grps, sgfrm contained almost double the number of unique contigs as sgfrs and triple that of sgfac. 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 relative to the other two libraries. a percent of total 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 contigs had similarities with different types of grp, based on published annotated sequences. contigs were considered to encode grps if the translated amino acid sequence contained a glycine content of at least 20%. the most abundant grp found among all three libraries was a amino acid protein containing 25% glycine obtained from r. haemaphysaloides and annotated as "unknown function". flagelliform silk proteins, identified from various spider species, was the second most abundant grp found among the three libraries. proteins annotated as cement and cement-like proteins from h. longicornis, i. scapularis and r. appendiculatus were also commonly observed among the three libraries. * computation of glycine content was calculated based on translated consensus sequence comparing the blast results of the three libraries shows that, with contigs representing ests, r. microplus contained the most abundant contigs homologous to grps as compared to contigs from a. cajennenes and contigs from r. sanguineus. salivary glands from r. microplus also contained the greatest variety of grp with contigs homologous to different grps whereas a. cajennense and r. sanguineus salivary glands contained and different grps, respectively. 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 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. these transcripts represented unique types of grps in r. microplus, whereas r. sanguineus and a. cajannense each presented only unique type of grp. on the other hand, only two grps were common to all three species of ticks and were represented by ests. we also analyzed the distribution of 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; r. microplus contains twice the amount of contigs encoding grps. a protein previously described in rhipicephalus haemaphysaloides haemaphysaloides, annotated as of "unknown function", represented the class of grp with which the majority of ests in the libraries presented similarity. over half of these transcripts derived from the library from r. microplus salivary glands. interestingly, the sgfrm library does not present any est with similarity to salivary gland-associated protein 64p from rhipicephalus appendiculatus ticks, at least on the first 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. 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, and salivary homologues are present in several species of ticks. 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 χ 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 more transcripts coding for all types of grps than the sgfac library; transcripts for grps were equally represented in the sgfrm and sgfrs libraries. 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. 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. 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 more transcripts of grps than the library from the salivary glands of the longirostrata, heteroxenous tick, a. cajennense. 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. *chi-square test for representation of the glycine-rich proteins distributed in the three cdna libraries. we also observed that the distribution of ests within some contigs was greater in a given species of tick. contig from sgfrm, coding for a protein similar to an "unknown" protein from r. h. haemaphysaloides ticks, was the most abundant transcript among the three libraries and the most abundant in the sgfrm library, with ests versus ests in both the sgfrs and sgfac libraries, however it was not differentially represented among the three ticks. a contig coding for a grp similar to "acanthoscurrin precursor" from acanthoscurria gomesiana spiders was also not significantly differentially represented, although sgfrs contains ests and sgfac has just est. however, the grp similar to "salivary gland-associated protein 64p" from r. appendiculatus, regarded as a cement protein, was significantly more expressed in salivary glands of female r. sanguineus than in those of a. cajennense. 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, and transcripts that were similar to silks of true spiders, 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 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 genes encoding secreted proteins increases in salivary glands of female ixodes scapularis ticks after attachment to their host and, interestingly, almost a third of these encode grps. furthermore, transcripts for grps were not observed in salivary glands from unfed females. kaufman 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, 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 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; the term "epidermis development" was most frequently assigned to transcripts from heteroxenous ticks. 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, a library made from a heteroxenous, brevirostrata tick. over half 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 ticks. phylogenetic analyses of glycine-rich proteins silk-like proteins phylogenetic analysis of grp contigs from the three ticks studied herein shows distinct clades as displayed in figure two of them contain a group of contigs that presented similarities with silk proteins of spiders. one was similar to a flagelliform silk protein,, which is a viscous, glue-like silk from orb-weaving spiders of the nephila genus; another was similar to major ampullate spidroin from kukulcania hibernalis, a cribellate, non-orb-weaving spider. interestingly, these clades are the most defined in the dendogram, as may be observed through the proximity among members of these clades. another feature of clades and is that each is formed by contigs from a single species of tick: clade presented contigs only from a. cajennense and clade contained contigs only from r. microplus, probably for this reason the clades showed better proximity among the members. this finding is compatible with our hypothesis that metastriate ticks presenting with different biological characteristics rely on different types of grps. noteworthy is the fact fsp is an elastic and glue -like adhesive silk, a characteristic which could be important for a brevirostrata monoxenous tick. the third distinct clade has showed similarity with an unknown protein from r. haemaphysaloides and contains contigs from only brevirostrata ticks, i.e., r. microplus and r. sanguineus, again a finding that is compatible with our hypothesis. futhermore, although distinct clades were not formed by the remaining grp contigs, we noted that contigs segregated into two different patterns: those which presented matches with spider silk proteins are concentrated in clades that are distant from those that have similarities with cement-like proteins of ticks, with the exception of contigs rs rm and ac this finding suggests that these two "major types" of grps may have different roles during attachment ticks on the host. the similarities found with spider grps were also with masps and fsps, albeit from different genera of spiders. interestingly, a. cajennense was represented only twice among the cement-like contigs, while two other cement-like contigs from this species grouped with spider silk-like contigs. a multiple alignment analyses of the three distinct clades showed the level of conservation among contigs, as can be observed in figure clade formed by contigs from sgfrm showed the greatest degree of conservation. sequence alignment of rm and rm show an identity of % and % similarity. rm and rm presented the lowest conservation, 33% identity and % similarity. contigs of clade formed by sgfac sequences also displayed low divergence, even the least similar transcript ac14; ac presented a reasonable equivalence between sequences whereas ac and ac had better conservation, presenting an identity of % and similarity of %. clade did not did not present the same level of conservation as clades and except for contigs rs and rm which presented a high identity and similarity of % and %, respectively. it was interesting to note the large conserved region visualized in each of the sequence alignments of clades and five conserved regions are encountered in sgfrm contigs of clade that are identical in the contigs: 1) qlgps, 2) sgslg, 3) gvlpsg, 4) sgvgrg and 5) tgfvlpg; some of these conserved regions could be extend if the charge and chemical proprieties of the residues from different contigs are taken into consideration. in the alignment for clade aspartic acid could change to glutamic acid at residue leucine to isoleucin at residue, glycine to serine at residue and valine to alanine at residue and these amino acids present similar characteristics among each other. the same aspects can be observed for residues 43- and 89- in addition, when the contigs of clade are compared with the composition of flageliform silk proteins, positions of important residues of silk proteins such as glycine, serine and proline are conserved among them. regarding clade two conserved regions are found in all sgfac contigs, one composed of residues, fgsgf, and a second one with residues, sglgggygsg. it is noteworthy that both regions have glycine and serine residues. again, conserved regions contain glycine and serine residues, two abundant amino acids in spider silk. alignment of ac contigs and masp proteins showed similarity in most positions containing glycine and serine residues, but not in positions with proline residues. sheets are formed in secondary structures of silk proteins with repeats containing glycine, serine and alanine, which confer their elastic and strength proprieties. the presence of a proline residue between serine and glycine, as happens in sequences of clade could be important to "interrupt" secondary structures determined by glycine, serine and alanine, promoting acquisition of more elastic and less stiff properties. the mechanical property of elasticity is greater in flagelliform silk proteins of orb-weaving spiders that are made to capture flying prey than in major ampullate spidroin silk proteins used in capture threads in less mobile spiders. the multiple alignments of clade sequences, which are homologous with an "unknown" protein from rhipicephalus haemaphysaloides did not present conserved regions, perhaps owing to divergence among contigs and many gaps that could not allow long conserved regions. however, it can be observed though shading of the alignments that they present similarities as described before, with regions abundant in glycine and serine. in addition to the contigs derived from r. microplus, r. sanquineus and a. cajennense analyzed herein and in order to increase the stringency of the test for our hypothesis, we performed a multiple alignment using contigs from the work of francischetti et al. that reviewed all of the available salivary components of ticks. this work described a superfamily of glycine-rich proteins for argasid and ixodid ticks. we observed in this work that argasid ticks produce only three types out of over four hundred types of grps. this maybe due to the fact that argasid ticks are rapid feeders and complete a blood meal in minutes. we also observed that the majority of the grps found in prostriate ticks, are collagen-like proteins. this group appears to have a primitive form of attachment among the ixodid ticks, presenting an intermediate complexity in this process. finally, this work showed that in metastriate ticks the grps belong to ggy, gyg and metastriate spider-like cement protein families. we therefore excluded analyses of grps from ixodes sp. and argasidae ticks and selected grps from the nr database on ncbi that present similarities with silk-like and cement-like proteins from a. variegatum, a. americanum, d. andersoni, r. microplus and r. appendiculatus. we aligned all sequences similar to silk-like proteins from our libraries and the nr database from sup-table and using the neighbor joining analysis produced the phylogram shown in figure sequences generated by the present work are symbolized with a circle, a square and a triangule. in addition to our sequences, other transcript sequences of other species ticks are represented: dermacentor andersoni and r. appendiculatus, both brevirostrata, heteroxenous 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 representatives of longirostrata, monoxenous ticks that could be included in this analysis. this approach showed that contigs from amblyomma ticks formed distinct clades. 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. all sequences from this clade present similarity with flagelliform silk. moreover, sequences in the dendogram displayed a wide-spread distribution, including a clade formed only by sequences from our libraries, showing that we have contributed 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. the cement-like sequences from the same species analyzed before were aligned and used to generate the phylogenetic tree shown in figure which showed six distinct clades. clades and contain cement-like proteins from other brevirostrata and longirostrata ticks, indicating the diversity among cement-like proteins. most sequences from clade presented similarity with the salivary gland-associated protein 64p from r. appendiculatus, except sequence rm which showed similarity with npl- from ixodes pacificus. clade contains a subclade with sequences derived exclusively from r. microplus. clade in turn, presented sequences similar to putative cement protein rim from r. appendiculatus and unknown protein from r. haemaphysaloides. clades and aside from illustrating the extensive diversity in the expression of cement-like proteins between the ticks, also indicate the expansion of the genus rhipicephalus showing similarity with cement-like antigen protein from haemaphysalis longicornis and an unknown protein from r. haemaphysaloides. finally, clade 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 vertebrate skin nucleic-acid-binding proteins and to the mycobacterium tuberculosis pe-pgrs multigene family. these similarities may also shed light on the biological functions of the tick grps. in plants many grps form the walls of initially polysaccharide-rich primary water pipes of elongating plant organs. 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 and similarly tick saliva can elicit local hypersensitivity reactions in immune hosts. grps also play a role in regulating permeability and penetration of toxins in insect cuticles. in ticks the cement cone may assist the cuticle 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, which are present in the local inflammatory infiltrate elicited in the host's skin by tick bites. this finding can also explain the significant quantity of transcripts from sgfrm 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 differentiated epithelial cells of vertebrate skin, may serve as decoys for the host. interestingly, the brevirostrata ticks herein analyzed displayed a greater number of transcripts related to development of epidermis and organization and biogenesis of extracellular matrix based on homologies to the go database than the longirostrata tick. finally, the products of the pe-pgrs multigene family of m. tuberculosis form a source of antigenic variation among different strains of this bacterium; 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 degradation in mycobacteria and epstein-barr virus. 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. grps present biochemical characteristics that could possibly be involved in stabilizing the tick to its feeding site for long periods due to their putative structural and mechanical functions inferred from the abundance of the amino acid glycine. grps may also block host immune system molecules that enter in contact with the tick mouthparts. many contigs were similar to silk proteins from spiders, such as fibroin, dragline, flagelliform, major ampullate spidroin and flag silks. each one of these fibers is composed of one or more proteins encoded by the silk fibroin gene family. spiders draw fibers from dissolved fibroin proteins that are stored in specialized sets of abdominal glands. it is interesting to note that ticks generate silk-like proteins from their salivary glands, while spiders use abdominal glands for this purpose and reserve their salivary glands for production of venom. tick silk-like grps may possibly support mechanical needs, as well as the capture of prey and predators. spider silks are being employed as scaffolds for engineering tissues and tick silk-like proteins may be more adequate for this purpose because of the intimate relation of this parasite with its host's skin. there are other precedents in nature for our finding that the distribution of distinct grps correlates with the biology of metastriate ticks. spiders, which are also arachnidae, offer a well known example: the architecture and mechanical properties of different spider webs are correlated with the biological characteristics of their spinners, for example, aerial versus terrestrial capture habits. these properties ultimately rely on the specialized functions of different types of silks. of interest to studies on the evolution of ticks, orb weaving by spiders is monophyletic, having evolved only once and speciation of spiders relates to use of different silks. genes encoding flagelliform silks were thought to be expressed exclusively by modern orb weaver spiders that make more elastic, gluey webs. however it was recently shown that cribellate orb weavers, which make drier webs, also express flagelliform silk genes, albeit in lower quantities. blackledge and colleagues suggested that an increase in the expression of flagelliform silk genes may have resulted in development of modern orb weavers. another example refers to the silks produced by salivary glands of simuliid filter-feeding flies. simulium noelleri and s. ornatum use silk pads to attach to substrates, the composition of which varies according the requirements of their habitats: s. noelleri feeds in lake outlets where weaker currents are found and s. ornatum feeds in open waters with stronger currents. accordingly, there are differences between ageing processes and biochemical composition of the silk pads from these two species, s. ornatum presenting the most durable structure. a third and final example is offered by larvae of two species of caddisflies. hydropsyche angustipennis spins hiding tubes and catching nets that collect food in water currents; larvae of limnephilus decipiens use silk fiber only for stitching fragments of grass into hiding and pupation cases. the composition of the silk fibers from these species differed by the arrangement of motifs in higher order repeats and by the presence of species-specific motifs. although the amounts of glycine are similar, the h-fibroin of h. angustipennis presents proline containing motifs, whereas l. decipiens presents a highly charged motif, eegrrr.  in the present work the differences observed for distribution of glycine-rich proteins were related to the number of hosts visited and to the anatomy of mouthparts of three species of metastriate ticks. all ixodid ticks, with the exception of some prostriate, present a strategy for attachment, but it differs among them. the species from the genus amblyomma, which belongs to the longirostrata ticks, secrete a casing around their long, fully inserted hypostome. in ticks from the brevirostrata group, which includes species from the genus rhipicephalus, the mouthparts are short and barely penetrate in epidermis, so a larger cement cone, from which grps have been purified, is necessary and is deposited in the upper layers of their host's skin. thus, it seems that the two brevirostrata ticks, r. microplus and r. sanguineus, need to express more glycine-rich proteins than the longirostrata tick, a. cajennense, in order to compensate for the small size of mouthparts and for the superficial fixation at the site of attachment. furthermore, r. microplus is monoxenous and r. sanguineus is heteroxenous and comparisons made between these ticks show that the former presents the greatest diversity of glycine-rich proteins, possibly because it is a one-host tick that feeds uninterrupted for many days until completion of its life cycle and, therefore, has greater demands for sustaining its attachment on host skin. contigs of salivary glands for several other species of ticks have also been examined. while the relative abundance of transcripts coding for glycine-rich proteins cannot be accurately compared between salivary gland libraries constructed in different laboratories and undergoing different biological situations, it is still noteworthy that annotation of the transcriptomes of salivary glands from female i. scapularis and i. pacificus indicate that prostriate ticks do not rely on glycine-rich proteins as heavily as metastriate ticks for their attachment to hosts or for other biological functions. on the other hand, salivary glands of females of d. andersoni, a metastriate, heteroxenous, brevirostrata tick, also contain abundant transcripts for grps: of the contigs containing the most abundantly expressed ests in salivary glands of females of d. andersoni, presented similarities to glycine-rich proteins and contained from to ests each. in conclusion, our findings furnish preliminary evidence to support the hypothesis that species of ticks with differences in the anatomy of their mouthparts and in the number of hosts they infest during their biological cycle rely on different types and quantities of glycine-rich proteins. this hypothesis must be further tested by expanding these observations to a larger number of species, by experimental approaches such as rna interference of expression of selected grps and by characterization of isolated grps. the data suggests that prostriate ticks rely on their elongated barbed hypostome mouthparts and make shallow cement cones, while the metastriate ticks rely on a larger and deeper cement cone possibly to compensate their relatively smaller mouth parts. the number of hosts visited by ticks during the parasitic stage of their life cycle also requires adaptations. according to balashov and hoogstraal and kim there was a transition from the three host to the two and one host cycle in hyalomma and in rhipicephalinae species of ticks. the biological characteristic of having a single host is regarded as an adaptation of this immobile ectoparasite to large nomadic animals since ixodid ticks die when they are unable to find a host. monoxenous ticks are thus better adapted to open environments inhabited by large, grazing ungulates. the ability to molt on the vertebrate reduces the number of necessary encounters and thus increases chances for tick survival. in addition to elucidating the biology of tick salivary proteins, the information contained in this work is relevant for the development of vaccines that target grps of ticks and that aim for protection against a broad range of species. the approach undertaken in this work can subsidize the choice of the different grps present in tick salivary glands for evaluation as protective antigens. 
ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. we found that different species of ticks rely on different types and amounts of grps in order to attach and feed on their hosts. these properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. metastriate ticks with short mouthparts express more transcripts of grps than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts. in this work, we analyzed expressed sequence tags similar to grps from cdna libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one or two or more, and the anatomy of their mouthparts, whether short or long. cdna libraries from salivary glands of the monoxenous tick, r. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, r. sanguineus and a. cajennense. transcripts of ests encoding grps were significantly more numerous in the salivary glands of the two brevirostrata species when compared to the number of transcripts in the longirostrata tick. the salivary gland libraries from brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders, whereas the longirostrata tick contained only contigs. to further investigate this relationship, we conducted phylogenetic analyses using sequences of grps from these ticks as well as from other species of brevirostrata and longirostrata ticks. these ticks were the monoxenous brevirostrata tick, rhipicephalus microplus, a heteroxenous brevirostrata tick, rhipicephalus sanguineus, and a heteroxenous longirostrata tick, amblyomma cajennense. glycine-rich proteins are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. the phylogenetic analyses of grps from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology.
 ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. glycine-rich proteins are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. these properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. in this work, we analyzed expressed sequence tags similar to grps from cdna libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one or two or more, and the anatomy of their mouthparts, whether short or long. these ticks were the monoxenous brevirostrata tick, rhipicephalus microplus, a heteroxenous brevirostrata tick, rhipicephalus sanguineus, and a heteroxenous longirostrata tick, amblyomma cajennense. to further investigate this relationship, we conducted phylogenetic analyses using sequences of grps from these ticks as well as from other species of brevirostrata and longirostrata ticks.  cdna libraries from salivary glands of the monoxenous tick, r. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, r. sanguineus and a. cajennense. transcripts of ests encoding grps were significantly more numerous in the salivary glands of the two brevirostrata species when compared to the number of transcripts in the longirostrata tick. the salivary gland libraries from brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders, whereas the longirostrata tick contained only contigs. the phylogenetic analyses of grps from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology.  we found that different species of ticks rely on different types and amounts of grps in order to attach and feed on their hosts. metastriate ticks with short mouthparts express more transcripts of grps than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts. 