BACKGROUND
Ædes ægypti is a highly anthropophagic and cosmopolitan mosquito vector of epidemic dengue and yellow fever.
to achieve fast blood feeding, adult female mosquitoes inject a complex salivary mixture into their hosts while probing for blood.
mosquito saliva, like that of other blood-feeding animals, has antihemostatic and antiinflammatory activities that counteract host responses that would otherwise restrict blood flow or call the attention of the host to the feeding site  <cit> .
a preliminary transcriptome of adult female salivary glands  has been previously reported  <cit>  where  <dig> full-length transcripts have been described based on an analysis of  <dig> expressed sequence tags .
of these putative proteins, ten have been verified by edman degradation of coomassie blue-stained bands from sodium dodecyl sulfate/polyacrylamide gel electrophoresis  of sg homogenates.
most salivary proteins found have no known function.
the genome of Æ. ægypti has been recently made available, facilitating further gene discovery.
in this paper, we present the analysis of an additional set of  <dig>  sg cdna sequences .
we describe  <dig> new transcripts,  <dig> of which code for proteins of a putative secretory nature, most of which have no known function.
we expect this work will contribute to the understanding of the evolution of blood feeding in arthropods and to the discovery of novel pharmacologic agents.
RESULTS
general description of the salivary transcriptome database
a total of  <dig>  clones was included in the est salivary database, including  <dig> previously described  <cit> .
the average length of the sequences was  <dig> bp, with  <dig> sequences containing a polya signature .
these clones assembled into  <dig>  clusters  and singletons .
in this paper, we will use the word 'contig' to refer to clusters of one or more sequences.
mitochondrial sequences, identified by their match to sequenced aedes albopictus and anopheline mitochondrial genomes, accounted for  <dig> est from  <dig> clusters .
of interest, several of these mitochondrial sequences mapped to scaffolds named supercont <dig>  and also to supercont <dig> , supercont <dig> , and supercont <dig> .
upon close inspections, these genomic scaffolds contain large segments of high similarity to ae.
albopictus mitochondrial genome.
accordingly, these contigs may be assigned to the mitochondrial genome in the final genome assembly, or may represent translocation of ae.
aegypti mitochondrial genes to the nuclear genome.
to attempt a functional classification of these unique sequences, we compared them with proteome databases by blastx and with protein motifs by rpsblast .
following manual annotation of these contigs, which included assignment of known or putative functions to the translation products, they were further divided into four categories: secreted  with  <dig> contigs and  <dig>  sequences; housekeeping  with  <dig> contigs and  <dig>  sequences; transposable element  with  <dig> contigs and  <dig> sequences; and a last category composed of contigs coding for proteins of unknown function  with  <dig> contigs and  <dig> sequences.
the unknown class may contain truncated transcripts mainly mapping to 3' untranslated regions of genes.
although the sclass corresponds to only 27% of the contigs, it consists of 64% of all est, reflecting the relatively low complexity and abundance of the secretory material of the sg, as indicated before  <cit> .
transcribed transposable elements
nine transcripts in our database possibly derive from transposable elements.
their translation products are similar to those of the sea urchin strongylocentrotus purpuratus <cit>  and to tc1-like transposase <cit> .
these transcripts may indicate active ongoing transposition activity in Æ. ægypti or, more likely, they may represent regulatory elements suppressing transposition of relatively recent genome invasions.
housekeeping gene products
putative hclass genes were further classified according to their possible function .
results are available online and can be searched on the columns labeled "class" and "comments"   <cit> .
more than 50% of the sequences in this class derive from transcripts associated with protein synthesis, energy metabolism, protein modification, and protein export.
transporters and signal transduction gene products are also highly represented in the library.
est matching transporter proteins were found for several v-type atpase subunits, na++k+ atpases, ca++ atpases, aquaporin, and several families of solute carriers.
v-type atpases have been implicated in the secretion of saliva in diptera  <cit> .
updated catalogue of salivary proteins
we used the transcriptome set   <cit>  and the artemis tool to identify novel proteins coded in the Æ. ægypti genome.
we have also assembled our transcripts with a set of ~ <dig>  est of Æ. ægypti available in public databases to obtain eventual full-length information of translation products.
this assembled est dataset, similarly organized as that for anoxcel  <cit> , is available online  <cit> .
using these tools, we identified  <dig> protein sequences, mostly full length, deriving from the salivary transcriptome of adult female Æ. ægypti mosquitoes.
of these  <dig> protein sequences,  <dig> were identified and contributed to genbank from this work, and the remaining were previously known.
of these  <dig> proteins, we identified a set of  <dig> putative secreted proteins expressed in the sg of Æ. ægypti,  <dig> of which are novel   <cit> .
to obtain additional information potentially useful to address future functional analysis, we determined the tissue and sex specificity of a selected subset of  <dig> transcripts encoding secreted proteins and corresponding to a total of  <dig> genes.
these  <dig> transcripts were selected based on their similarity to transcript families found in diverse mosquito species, and in the presumption that their translated products might play a role either in sugar or blood feeding.
oligonucleotide primers suitable for amplification of corresponding mrna were employed for reverse transcriptase-polymerase chain reaction  amplifications using as template total rna extracted from female sg, female carcasses , and whole adult males.
primers amplifying the ribosomal protein s <dig> were used for normalization and as control.
the results of this analysis are summarized in table  <dig>  we previously used a similar assay for the analysis of the anopheles gambiæ salivary transcriptome  <cit>  and obtained results overlapping very well with the information independently obtained by marinotti et al.
<cit>  using the affymetrix microarray chip.
the results reported here also fit well with the data obtained comparing salivary versus nonsalivary libraries   <cit> .
with our assay, it is possible to distinguish three broad classes of genes.
first, genes that are female sg specific or whose expression is enriched in the female glands: they are indicated as sg or enr, respectively, in table  <dig>  products encoded by these genes are likely to play some role in blood feeding, for example as antihemostatics or immunomodulators.
among those genes analyzed,  <dig> belong to this class; more precisely,  <dig> were female gland specific, and  <dig> were enriched in female sg.
they include both genes with unknown functions and genes known from previous studies on other mosquito species to be involved in the acquisition of blood meals .
a second group is represented by genes expressed in female glands and in adult males, without any expression in female carcasses: these are identified as sg,m in table  <dig>  it is very likely that most of these genes are gland specific and expressed both in male and female glands.
the corresponding products may be involved in sugar feeding, antimicrobial activity, or other gland functions;  <dig> transcripts are members of this group.
finally, the last class includes genes with ubiquitous expression, i.e.
expressed at approximately the same level in the three tissues examined and indicated as ubiq in table  <dig>  these genes most likely encode polypeptides involved in housekeeping functions:  <dig> of the transcripts analyzed belong to this group.
the following is a detailed description of the full-length transcripts found in the sg of adult female Æ. ægypti and of their profile of expression.
the four classes are sg ; enr ; sg,m ; and ubiq .
clones marked with # were analyzed three times, all others twice.
clones  <dig>   <dig>  and  <dig> may undergo alternative splicing.
although clones  <dig> and  <dig> ,  <dig>   <dig>  and  <dig>  were assigned to sg class, some faint bands were seen in some samples from males.
clones  <dig>   <dig>   <dig>  and  <dig>  underwent  <dig> cycles due to low amplification levels.
secreted salivary proteins
proteins with some function confirmed or presumed from structure
secreted ligand carrier-like proteins including d <dig> family
d <dig> salivary proteins
the first d7-coding gene was reported  <dig> years ago, for the mosquito Æ. ægypti  <cit> .
it was later found in virtually all mosquito sialotranscriptomes where short  and long  forms were recognized  <cit> .
in an.
gambiæ, this gene family, except for one poorly transcribed gene, appears to be selectively expressed in the female sg  <cit> , indicating a role in blood feeding.
this protein family is distantly related to the odorant-binding protein  family, which specializes in binding small ligands  <cit> .
recently, some mosquito d <dig> proteins were shown to bind and inhibit the action of biogenic amines such as serotonin, histamine, and norepinephrine, a function that might help blood feeding  <cit> .
additionally, one short d <dig> protein from anopheles stephensi, named hamadarin, was shown to prevent kallikrein activation by factorxiia  <cit> .
long d <dig> forms also exist in sand flies  <cit>  and culicoides  <cit> , indicating that this gene family was recruited very early in the evolution of hematophagous nematocera.
in the mosquito an.
gambiæ, five short and three long d <dig> proteins are known.
their genes are organized in a inverted tandem repeat <cit>  where the coding region for the three long proteins is followed by the five genes coding for the short protein in the reverse orientation  <cit> .
in Æ. ægypti, three short and two long d <dig> proteins map to the assembled genome supercontig <dig>  , and one short d <dig> protein maps to supercontig <dig>  .
the genomic region coding for the d <dig> proteins in supercontig <dig>  shows three short d <dig> genes followed by two genes coding for long forms; however, while in anopheles the frame orientation of the short and long forms is the same , in Ædes there is no consistent orientation .
similarly to an.
gambiæ, all large Ædes d <dig> genes contain five exons ; however, all short Ædes d <dig> genes have two exons , including that in supercontig <dig>  , while in anopheles, four of the five genes coding for short d <dig> proteins have three exons.
the anopheline gene coding for the two-exon gene is poorly expressed, leading to the suggestion that this two-exon gene may be turning into a pseudogene  <cit> .
the differences between anopheles and Ædes in the number of d <dig> coding genes, their exon number, their orientation, and their chromosome location  are consistent with the ~ <dig> million years of separation between culicines and anophelines  <cit> .
in agreement with the larger genome size of Ædes  <cit> , the five-gene d <dig> cassette of Ædes spans nearly  <dig> kb, four times more than the eight-gene cassette of anopheles.
to investigate whether additional genes were expressed within the d <dig> gene cassette in Æ. ægypti, we mapped ~ <dig>  est to the Æ. ægypti genome.
no new identifiable expressed genes were revealed in the d <dig> region; however, in the vicinity of the short d <dig> cassette, we found two transcripts, both deriving from sg libraries, which map to the intron of d7s <dig> and to the 3' region of the same gene .
translations of these two est do not reveal extended open reading frames.
this finding is reminiscent to the d <dig> short gene region of anopheles, which also has an apparently noncoding est mapping to the end of the short cassette, but at its 5' end.
we have hypothesized previously that these noncoding transcripts could be associated with transcriptional regulation of the cassette.
alignment of the d <dig> sequences from Æ. ægypti with those of Æ. albopictus, culex quinquefasciatus, an.
gambiæ, and one d <dig> salivary protein from the sand fly lutzomyia longipalpis indicates, as shown before, that the short d <dig> proteins appear to be truncated versions of the long d <dig> proteins, which appear to be the ancestral type .
the resulting phylogram, using the sand fly sequence as an outgroup, shows three clades without strong bootstrap support; however, the inner tree branches show considerably more conservation of the multiple forms within genus than between genera.
for example, the common long d <dig> clade  shows that two of the long d <dig> proteins of an.
gambiæ are more closely related to each other as are the culex or the Ædes pair.
within the Ædes genus, the Æ. albopictus and Æ. ægypti homologues are distinctly grouped together, indicating that they share a relatively recent common ancestor before the duplication event, as expected from these two mosquitoes of the same subgenus.
the same pattern is visible in the culicine short d <dig> clade  where all short d <dig> proteins are more related to each other within genus than between genera.
this is even more remarkable in the short d <dig> proteins of anopheles, where all short d <dig> proteins form a single clade outside of their culicine counterparts.
if the gene duplication events that lead to the formation of long and short d <dig> proteins occurred in the primordial mosquito ancestral to both culicines and anophelines, the tree pattern observed would be one where the orthologous pairs would be more similar to each other between genera than within genus.
two possible explanations may account for the observed tree pattern: either the gene duplications leading to the d <dig> expansions occurred independently after the division of the culicine and anopheline lineages, or some degree of gene conversion occurred within each species, maintaining the uniformity of the genes within species.
this latter scenario is consistent with the proposed primordial role of the d <dig> proteins, e.g.
sequestration of host serotonin released by thrombocytes at the site of bite, a function that would require the d <dig> proteins to be a major salivary protein constituent  <cit> .
the gene duplication event would be beneficial in allowing increased transcript mass needed to create the substantial amount of protein needed in the mosquito saliva to chelate the near-micromolar concentration of the vasoactive amine.
gene conversion events to maintain this function on multiple genes could be beneficial at this earlier stage of blood feeding evolution.
this phenomenon would maintain intraspecific copies of the gene family more similar to each other than to the orthologous interspecific copies.
with time, other salivary proteins may have taken a similar role of preventing platelet function, allowing the d <dig> proteins to acquire different functions such as binding other amines or to become anti-bradykinins, a function apparently only acquired in the anophelines, which diverged from the culicines ~ <dig> million years ago  <cit> .
for a review on the evolution of gene families, see references  <cit> .
evidence for synthesis of the two large d <dig> proteins and for d7s <dig> was shown before from edman degradation results of sds-page gels  <cit> .
presently, we observed extensive coverage of tryptic fragments for both long d <dig> proteins  as shown by two-dimensional  gel electrophoresis   <cit> .
this protein family appears polymorphic.
the predicted translation products of some of these alleles are shown in additional file  <dig>  as expected from this protein family, all transcripts described in additional file  <dig> are more expressed in the sg cdna libraries than in the remaining libraries, three of which are significant by the χ <dig> test at the  <dig>  level.
rt-pcr experiments agree with these results, indicating a selective or preferential expression of this gene family in female sg .
it should be noted, however, that transcripts encoding the short d <dig> were exclusively found in female glands, whereas mrna encoding the long d <dig> were also detectable at a lower level in adult males  and in other female tissues .
this observation may be connected to an independent regulation of the short and long d <dig> cassettes, which are more that  <dig> kb apart.
phosphatidylethanolamine binding proteins
we report three members  of this very ubiquitous protein family never found before in sialotranscriptomes of blood-sucking arthropods  <cit> , two of which have clear signal peptide indicative of secretion.
this protein family is known to bind lipids and was also shown to have serine protease inhibitory capacity.
their role in saliva is unknown.
no enrichment was found for these transcripts on the sg libraries when compared with other libraries.
other small molecule binding proteins
an obp and a lipocalin with a juvenile hormone binding motif were found in the sialotranscriptome of Æ. ægypti, both containing distinct peptide signal indicative of secretion.
the d <dig> proteins belong to the obp superfamily.
lipocalins are abundantly expressed in tick and triatomine sialomes, where they act as nucleotide -and biogenic amine-binding proteins in addition to other functions.
their function in Ædes is unknown.
no enrichment was found for these transcripts on the sg libraries when compared with other libraries.
secreted protease inhibitors
serpins
two serpins have been described before in Æ. ægypti, one of which has been characterized as an inhibitor of factorxa of the clotting cascade  <cit> .
we present one allele of the fxa-directed anticoagulant precursor having only 89% identity to the reported protein <cit>  which originated from the rockefeller strain of Æ. ægypti, and two alleles of a novel salivary serpin mapping to supercontig <dig> .
the three genes coding for these serpins are not located near each other in the Æ. ægypti genome.
all three salivary Æ. ægypti serpins have corresponding homologues found in ae.
albopictus sialotranscriptome <cit> .
the novel serpin has abundant tryptic fragment matches recovered by proteomics , indicating its expression in the sg, as did gi| <dig> .
transcripts for all serpins are significantly overrepresented in the sg library when compared with the remaining libraries, in accordance to the rt-pcr experiments shown in table  <dig>  which indicates that two of the three serpins are female specific and that one may be found also in males but not in carcasses of females not containing sg .
other protease inhibitors
a kazal domain-containing peptide, similar to one described in ae.
albopictus and to several other proteins described as thrombin inhibitors, was found in the Æ. ægypti sialotranscriptome.
a cystatin was also found, but this protein is reported as truncated; we were not successful in searching the genome for the missing exon.
this could be due to the large intron size observed in ae.
aegypti.
accordingly, no indication of secretion is possible, but it is described in this section due to the importance of this family in inhibiting proteases associated with inflammation.
both transcripts are ubiquitously found in mosquito tissues by rt-pcr and may play a housekeeping role.
vasodilator
sialokinin
the gene coding for this endothelium-dependent peptide vasodilator  <cit>  has been reported earlier and shown to be transcribed specifically in female sg  <cit> .
although two forms of the peptide have been described earlier differing in the aminoterminal , only one gene is found coding for this peptide sequence, for which  <dig> est were found in the salivary cdna library.
the asn version may have been an artifact from the modification of the original peptide, which was stored in acidic solution.
enzymes
nucleotidases
the salivary purinergic degradation machinery of Æ. ægypti comprises the enzymes apyrase , adenosine deaminase , and purine hydrolase  <cit> , which may serve an antihemostatic and antiinflammatory function by removing nucleotide agonists of platelet aggregation and mast cell degranulation.
in addition to these previously described enzymes, we found a second 5' nucleotidase that may function either as an alternative apyrase or as a secreted salivary 5' nucleotidase, as is the case with lutzomyia longipalpis  <cit> .
the novel 5' nucleotidase has only 38% identity to the previously characterized apyrase form of aedes aegypti <cit>  but has a higher identity  to a culex.
quinquefasciatus salivary 5' -nucleotidase/apyrase protein <cit> .
5' nucleotidases are typically seen in the external part of the cellular membrane to which they are bound by a inositol phosphate anchor  <cit> .
secreted apyrases and 5' nucleotidases have lost either the conserved ser or the surrounding lipophylic amino acids   to which the inositol phosphate moiety binds to  <cit> .
the novel Ædes 5' nucleotidase, like the previously described salivary apyrase  <cit> , lacks the typical ser residue surrounded by hydrophobic aa typical of membrane-bound enzymes, similarly to other mosquito salivary 5' nucleotidase , supporting their role as secreted 5' nucleotidases.
this novel apyrase may contribute to the purinergic degradation machinery found in saliva of Æ. ægypti.
all these genes are overrepresented in sg libraries and, except for ada, significantly so.
rt-pcr results are somewhat contradictory with the proposed role of these enzymes in blood feeding: with the exception of the ada coding transcript that was enriched in female sg, the other genes appeared to be sg specific, because they are expressed in sg of females and in whole males, which would suggest a role in sugar feeding, instead.
we do not have a good explanation for this observation; however, we should point out that apyrase, purine nucleosidase , and ada showed very similar expression profiles by rt-pcr in the related mosquito Æ. albopictus .
evidence of synthesis of these enzymes was found for the ada, the original apyrase, and the pnase, which provided abundant tryptic fragments .
a novel ribonuclease of the t <dig> family <cit>  was also characterized.
this enzyme has not been previously characterized in sialotranscriptomes.
it has a typical signal peptide indicative of secretion and may function in the degradation of extracellular rna  <cit> .
serine proteases
nine secreted serine proteases varying in predicted mature molecular weight between  <dig> and  <dig> kda were found in the Æ. ægypti sialotranscriptome, seven of which are being reported for the first time .
two of these serine proteases  contain a cub domain <cit> , indicating specialized substrate recognition.
both are found in supercontig <dig>  within  <dig> kb of each other.
some of these enzymes  are possibly related to immunity and are similar to other enzymes annotated as prophenoloxidase activators, but they could have been co-opted to function in hydrolyzing specific host proteins.
a smaller number of this type of enzymes found in an.
gambiæ sialotranscriptomes was selectively expressed in the sg of adult females, indicating they may play a role in blood feeding, perhaps by activating antiinflammatory pathways  or deactivating inflammation.
one such Ædes enzyme is ae- <dig> <cit> , similar to proteins annotated as chymotrypsin, which is overexpressed in Ædes sialotranscriptomes as opposed to the remaining transcriptomes.
three of these serine proteases were significantly underrepresented in the nonsalivary-gland libraries.
four serine proteases tested by rt-pcr agreed with the library frequency results: the transcripts were found in female glands and adult males but not in female carcasses deprived of sg.
sugar hydrolases
previously reported amylase <cit>  and alpha-glycosidase/maltase <cit>  are abundantly overrepresented in the salivary est collection.
these genes were shown to be expressed in the proximal regions of the female glands, the region associated with sugar feeding.
other hydrolases
an alkaline phosphatase <cit>  and a carboxylesterase <cit> , both containing signal peptides indicative of secretion, are described.
both enzymatic activities in adult female sg have been previously described in Ædes  <cit> , and the esterase activity shown in saliva, but their function in blood feeding can only be speculated upon.
immunity-related proteins
the sg of mosquitoes produce various antimicrobial polypeptides and other immunity-related products such as bacterial surface-recognizing proteins and lectins that may be important in opsonization and initiation of activation of the ppo enzyme leading to pathogen melanization.
the purpose of these products may be to control microbial growth in the sugar solutions stored in the crop or in the gut following a blood meal.
previously, lysozyme <cit>  was found in both male and female sg of Æ. ægypti, and in the mosquito crop  <cit>  and shown to be secreted by females following either a sugar or blood meal  <cit> .
indeed, Æ. ægypti salivary lysozyme is significantly overrepresented in the sg libraries, and the rt-pcr experiment supports salivary expression in both male and female sg .
anopheline mosquitoes also display a similar pattern of lysozyme expression in the proximal gland region  <cit> .
it is possible that most immunity-related gene products follow the same pattern of expression shown by lysozyme.
some of the enzymes possibly associated with ppo activation have been listed above in the enzyme section.
c-type lectins
five c-type lectins are described in additional file  <dig>  four of which are novel.
expression of ae- <dig> and gi| <dig> was confirmed by mass spectrometry following tryptic digestion of protein bands .
these two lectins are also expressed significantly more in sialotranscriptomes than in other Æ. ægypti cdna libraries, indicating that they are possibly salivary-tissue specific.
in accordance with this expectation, rt-pcr experiments indicate expression exclusively in female sg .
the two genes coding for these salivary-specific lectins were found as an inverted tandem repeat in supercontig <dig> , each with a single intron separating the signal peptide gene region from the remaining coding sequence.
two other c-type lectins tested by rt-pcr were ubiquitously expressed.
the c-type lectin family is expressed in most mosquito sialotranscriptomes described thus far.
this protein family is implicated in immune recognition phenomena in general and in plasmodium development in anopheles in particular  <cit> .
despite these not-yet-demonstrated roles of c-type lectins in salivary immunity, it is interesting that in snake venoms this protein family has been recruited to perform various unrelated functions such as anticlotting, toxin, and platelet aggregation inducer  <cit> .
lectins may also play a role in the colectin pathway of complement activation  <cit> .
hemagglutinins were described in anopheline sg more than  <dig> years ago  <cit> .
this activity may help concentration of red blood cells in the mosquito gut  <cit> .
the molecular nature of any anopheline hemagglutinin, however, is unknown.
differently from anophelines, and despite having salivary lectins, Ædes sg homogenates lack hemagglutinins, indicating that the salivary lectins do not recognize vertebrate red blood cells or that they are monomeric in their carbohydrate binding site.
overall, it appears that the two female sg-specific lectins may have a role in hemostasis rather than immunity.
other proteins with sugar-binding domains
aet- <dig> and aet- <dig> are similar to, but shorter than, n-acetylgalactosaminyltransferase and glucuronyltransferase, respectively, appearing to derive from novel genes that arose from gene duplications and partial deletions of ancestral genes coding for carbohydrate binding enzymes, the final products lacking the original carboxyterminal domain.
aet- <dig> has a partial pfam glycos_transf_ <dig> motif that comprises a diverse family transferring sugar from udp-glucose, udp-n-acetyl-galactosamine, gdp-mannose, or cdp-abequose to a range of substrates including cellulose, dolichol phosphate, and teichoic acids.
aet- <dig> has a weak match to the pfam udpgt motif and is similar to proteins in the nonredundant  database annotated as udp-glucosyl transferase.
it is possible that these proteins have a destination in the endoplasmic reticulum or golgi and do not have a secretory nature.
their function is unknown.
the proteins annotated as imaginal disk growth factor protein  <dig> <cit>  and aea-871bre were expressed in the sialotranscriptome of Æ. ægypti.
these proteins have a chitinase domain and are homologous to an.
gambiæ bacteria responsive protein  <dig> <cit>  and bacteria responsive protein  <dig> <cit> , which were shown to be immune-responsive chitinase-like proteins that have lost chitin-binding activity  <cit> .
angiopoietins/ficolins
this group of proteins has the pfam fibrinogen c motif <cit> seen in invertebrate proteins displaying lectin activity toward n-acetylglucosamine residues and implicated in immune function  <cit> .
in an.
gambiæ, the ficolin family was expanded in comparison to drosophila melanogaster, where  <dig> members were seen in its genome as opposed to  <dig> in the fruit fly  <cit> .
three proteins belonging to this family are shown in additional file  <dig>  two of which are novel.
evidence for salivary expression of gi| <dig> and ae- <dig> was found by mass spectrometry in tryptic digests of protein bands .
of interest, the two genes for these proteins occur as a tandem repeat in supercontig <dig> .
est for these two genes are also overrepresented in the sialotranscriptomes and are indicated to be female-salivary-gland specific by rt-pcr experiments, thus suggesting a blood-feeding rather than an immune role for these proteins.
antimicrobial peptides
the gene products for the amp gambicin <cit> , lysozyme <cit> , and defensin a <dig> <cit>  have been previously described in Æ. ægypti sialotranscriptomes and are listed in additional file  <dig>  transcripts encoding gambicin and defensin a <dig> were detected by rt-pcr in all tissues examined, indicating ubiquitous expression ; however, a significant overrepresentation of the corresponding est in sialotranscriptomes should be pointed out.
we additionally describe three novel peptides that may have an antimicrobial function.
aet- <dig> <cit>  has gy repeats that are also found in peptides of similar size known to have antimicrobial activity in nematodes  <cit> .
aet- <dig> <cit>  and aet- <dig> <cit>  are candidate amp containing a hhh motif seen in other histidine-rich amp  <cit> .
aet- <dig> appears to be sg specific, as a total of  <dig> est was found in the combined sg transcriptome, although none were seen in other tissue transcriptomes.
rt-pcr confirmed the presence of the transcript in female glands and male bodies but not in female carcasses without sg .
other immune-related gene products
a peptide  closely related to a previously described Æ. ægypti peptide named i23r <cit>  potentially involved in plasmodium susceptibility  <cit>  was found in the sialotranscriptome.
we also present an allele to aea-233a, indicating the polymorphism of this gene.
the Æ. albopictus sialotranscriptome revealed a homologue that is 63% identical, but no significant matches were found to any other animal or plant proteins in the nr database.
this peptide may belong to a not-yet-characterized antimicrobial gene family specific to the Ædes genus.
expression of aea-233a was ubiquitous by rt-pcr.
two other gene products are described, both associated with pathogen surface-pattern recognition: the previously described gram negative binding protein <cit> , which is significantly overrepresented in sialotranscriptomes and appears expressed both in female sg and in adult males , and the novel ae- <dig> <cit> , which is similar to peptidoglycan recognition proteins and was ubiquitously expressed by rt-pcr experiments.
mucins
mucins and peritrophins are proteins associated with lining of epithelia or inert extracellular structures, such as chitin.
mucins are highly glycosylated proteins containing ser or thr modified with n-acetylgalactosamine residues.
their expression in the sg may have a function of lining the chitin surfaces of the mouthparts, but they may also assist in antimicrobial functions.
we present  <dig> mucins in additional file  <dig>   <dig> of which are novel, including one allele.
these proteins have an average ser+thr equal to  <dig> % of their total aa, as opposed to  <dig> % observed as the average of all proteins found in additional file  <dig>  we additionally report on a polypeptide  containing three glycosylation sites and one chitin-binding domain, which may be involved in proximal lining of the cuticular duct.
all other proteins have 11– <dig> glycosylation sites.
putative secreted proteins without functional classification
belonging to ubiquitous protein families
antigen <dig>  family
ag5-related salivary products are members of a group of secreted proteins that belong to the cap family   <cit> .
members of this protein family are found in the sg of many blood-sucking insects  <cit> .
most of these animal proteins have no known function; in the few instances to the contrary, they diverge from proteolytic activity in conus  <cit> , to smooth muscle-relaxing activity  <cit>  in snake venoms, to salivary neurotoxin in the venomous lizard heloderma horridum  <cit> .
three members of this gene family were previously described in the sialotranscriptome of Æ. ægypti.
est's for all three genes are overrepresented in the sialotranscriptome as compared with the combined transcriptomes, indicating they may be preferentially expressed in the sg.
in accordance with these results, gi| <dig> was exclusively transcribed in female glands as indicated by rt-pcr, suggesting an antihemostatic function for the gene product, while the other two genes are transcribed in female glands and male bodies but not in female carcasses.
differently to an.
gambiæ, which has four salivary ag <dig> members, three of which cluster in chromosome arm  <dig> l  <cit> , the salivary ag <dig> proteins of Ædes do not appear to cluster in the genome, mapping to different supercontigs.
other secreted proteins of unknown function found in non-bloodsucking insects
eight putative secreted proteins have similarities to proteins or protein domains found in non-bloodsucking insects.
one of these proteins   <cit>  is a truncated fragment where it is not possible to identify whether it has a signal peptide indicative of secretion, but it has a weak cdd domain pan_ap_hgf <cit> , which is found in plasminogen/hepatocyte growth factor proteins, and various proteins found in bilateria, such as leech antiplatelet proteins; however, the mrna encoding this protein was found ubiquitously expressed by rt-pcr and may not have a unique salivary role in blood feeding.
ae- <dig> <cit>  has a til <cit>  domain  and is significantly overexpressed in sialotranscriptomes.
rt-pcr indicates both male and female sg may be the target tissue of expression of this peptide .
peptides containing a til domain were also found in the an.
stephensi <cit>  and an.
gambiae adult male sialotranscriptomes <cit> .
the finding of this type of peptide being overexpressed in male an.
gambiæ sg indicated a possible antimicrobial function rather than a function as a host serine protease inhibitor during blood feeding.
indeed, a tick til domain containing peptide named ixodidin <cit>  was found to have an antimicrobial function in addition to inhibiting serine proteases  <cit> .
the remaining six polypeptides have similarities to drosophila or other species, and their structure does not hint at any particular function.
belonging to families only found in blood-sucking diptera
56-kda family
this protein family has been found to date only in salivary transcriptomes of adult mosquitoes, including adult male an.
gambiæ.
the sg specificity of this gene transcript in Æ. ægypti is supported by significant overrepresentation of est on the sialotranscriptome and by rt-pcr .
all family members have a signal peptide indicative of secretion and a predicted molecular weight near  <dig> kda.
blast comparisons <cit>  also show weak similarity to bacterial proteins but to no other eukaryotes.
following  <dig> iterations of psi-blast <cit> , only mosquito and bacterial proteins are retrieved  <cit> , suggesting that this family of proteins may have originated as a lateral transfer from a bacterial genome to the ancestral mosquito genome.
the single exon structure of the gene <cit>  – unusual in eukaryotes, particularly for a protein of this size, but the rule in prokaryotes – supports this hypothesis.
the an.
gambiæ homologue also displays a single exon gene structure  <cit> , as reported previously  <cit> .
the bacterial proteins retrieved by psi-blast are mostly annotated as phage-associated proteins, suggesting the lateral transfer might have occurred via a phage-associated mechanism.
41-kda family
two novel alleles coding for this protein family are described in additional file  <dig>  the gene coding for this protein has a  <dig> exon structure <cit>  on supercontig <dig> .
the gene product shows similarities to proteins of comparable size found in Æ. albopictus and culex pipiens and to a shorter protein in salivary transcriptomes of culicoides sonorensis.
a match to Æ. ægypti gi| <dig> <cit>  probably represents a misannotated protein with a dna frame-shift error.
three iterations of psi-blast <cit>  only retrieved culicine and culicoides proteins, indicating the uniqueness of this protein family.
no salivary anopheline sequences similar to the 41-kda family have been reported, including all ensembl-predicted an.
gambiæ proteins deposited at the national center for biotechnology information  .
est coding for the two alleles in Æ. ægypti are significantly underrepresented in the non-sg libraries; rt-pcr suggests ubiquitous expression of one of the alleles, although only very faint bands were detected even after  <dig> cycles of amplification .
the function of any member of this protein family is unknown.
30-kda ge-rich family
this acidic, gly/glu-rich protein family is abundantly expressed in adult female mosquito sg, where they appear to be involved in allergic reactions to mosquito bites  <cit> .
in Æ. ægypti, two proteins of this family have been previously reported, and we now report two additional splice variants and alleles.
evidence for expression was found in bands labeled  <dig> ag  in the 2d gel experiment shown in figure  <dig>  the two proteins are coded by an inverted tandem repeat in supercontig <dig>  separated by only  <dig> base pairs.
the sialotranscriptome is significantly overrepresented in est coding for this protein family, indicating it is salivary specific.
rt-pcr confirms the female sg specificity of these transcripts.
based on the public sequences available, it appears that in anopheline mosquitoes , only one polymorphic gene exists for this protein family per genome.
29-kda family
two different transcripts <cit>  in Æ. ægypti are possibly obtained from the same genomic region coding for the basic  salivary protein ae- <dig> and for the alternative shorter transcript ae-236a, which was not found on the salivary est but rather as one contig assembled from four est deriving from nonsalivary libraries <cit> .
blast comparison of the deducted salivary protein with the nr database shows similarities to other culicine and anopheline salivary proteins, including weak similarities to some members of the  <dig> kda protein family.
four iterations of psi-blast <cit>  are able to assemble only salivary proteins of mosquitoes, culicoides, and phlebotomus, including all 30-kda proteins discussed above, suggesting that either this unique protein family was co-opted as salivary proteins independently by these different families of diptera or that they have a common blood-feeding ancestor.
clustalw alignment of the sequences shows that following the signal peptide region, a subset of the proteins have a ser/thr/gly-rich region, poor in aliphatic aa as shown in figure  <dig> by the richness in brown residues.
the carboxyterminal region is marked by the alternation of polar and aliphatic residues.
a subset of culicoides proteins does not have this domain.
the phylogenetic tree shows a robust mosquito clade  with three members of the family per Ædes species  and one member  per anopheline species, which have only a single 30-kda member.
a single cx.
pipiens sequence is also part of this clade.
cladeii has two very similar culicoides proteins.
cladeiii has very divergent culex and phlebotomus sequences, and cladeiv has solely culicoides sequences, representing those lacking the ser/thr/gly-rich aminoterminal domain.
it is tempting to speculate that these data support a common origin of blood feeding for these three dipteran families, where two genes are found in culicines, a single in anophelines and possibly phlebotomus, and a rather large gene expansion in culicoides, which has at least seven genes in the family.
notice that cladeiii, containing mosquito and sand fly sequences, roots with culicoides cladeiv and that culicoides cladeii roots with the mosquito cladei, indicating that culicoides may have shared a common ancestor with mosquitoes that had two genes of this unique family.
alternatively, convergent evolution may have shaped these genes to produce similar proteins.
ae-236a was enriched in the sg of adult females, while ae- <dig> was significantly underrepresented in non-sg libraries, suggesting a salivary specificity for this protein family, as is the case with the related 30-kda family discussed above.
other mosquito-or diptera-specific peptides
additional file  <dig> also includes  <dig> additional polypeptides, one of which is an allele, showing sequence similarities to putative proteins from other hematophagous diptera including, in a few cases, some weak similarities to drosophila;  <dig> of these are novel.
among this class of polypeptides, nine were analyzed by rt-pcr , and seven are significantly underrepresented in the non-sialotranscriptomes, as follows: ae- <dig> <cit> , which is similar to drosophila and culicoides proteins of unknown function and was ubiquitously expressed by rt-pcr experiments; two alleles  coding for basic  29-kda proteins containing  <dig> putative galactosylated ser/thr and similar to culex and culicoides <cit>  salivary proteins; three polypeptides whose expression appeared gland specific, as suggested by rt-pcr .
ae- <dig> , is weakly similar to proteins varying in size from 150–180aa residues from anopheles <cit>  and drosophila <cit> .
two additional transcripts, ae- <dig> and ae- <dig>  were expressed solely in the adult female sg by rt-pcr.
the function of these proteins remains to be investigated.
genes belonging to protein families found to date only in Ædes genus
nineteen genes were found expressed in the sialotranscriptome of Æ. ægypti coding for polypeptide families known only in the Ædes genus, as follows:
62-kda family
two single exon <cit>  genes separated by ~ <dig> kb in supercontig <dig>  code for proteins with signal peptides and mature mass of 62– <dig> kda.
transcripts for these genes are significantly overrepresented in the sialotranscriptome and are shown to be adult female sg specific by rt-pcr .
they are similar to homologous salivary protein sequences seen in ae.
albopictus <cit>  and, to a much smaller degree, to rhoptry proteins of plasmodium.
repeated leu and glu residues provide similarities to myosin <cit> , indicating this protein family may be involved in adhesion phenomena.
their uniqueness among metazoan and single-exon structure indicates possible horizontal acquisition of this gene family in Ædes.
both genes are abundantly expressed in the sg as evidenced by bands labeled  <dig> k by 2d gel electrophoresis ms/ms .
34-kda gene family
seven transcripts coding for related proteins were found mapping to supercontig <dig> .
after locating the corresponding genomic regions, these  <dig> transcripts were annotated as truncated forms or alleles of three genes found as a tandem repeat .
we additionally found one possible related gene in the most distal region of the 34-kda cassette .
except for the first gene on the cassette, which codes for a 16-kda protein and has two exons <cit> , the remaining genes are single exonic <cit>  and code for proteins of ~ <dig> kda.
the two central genes, each with a single exon, are abundantly expressed as evidenced by ms/ms sequencing of tryptic digested bands .
all transcripts matching this gene region are significantly overexpressed in the sg transcriptome when compared with the remaining libraries.
rt-pcr indicates they are enriched or exclusive of adult female sg .
protein products of these genes match significantly only ae.
albopictus proteins <cit> .
psi-blast for each transcript against the nr protein database reveals cytoskeletal proteins such as actin and myosin, mainly due to the presence of repeated charged aa.
this indicates that this protein family may be associated with adhesion phenomena .
the single-exon nature of most members of this gene family and their uniqueness among metazoa points to a horizontal acquisition of this gene.
<dig> -kda family
two genes coding for proteins of ~ <dig> -kda  are found as a tandem repeat on supercontig <dig> .
the gene coding for gi| <dig> is abundantly expressed as evidenced by ms/ms of tryptic digested band labeled  <dig>  in figure  <dig>  these proteins are similar only to homologues <cit>  found in ae.
albopictus <cit> .
both genes are significantly overtranscribed in the sialotranscriptome when compared with other transcriptomes.
rt-pcr indicates enrichment in the female sg or exclusive expression in the same organ.
9-kda family
two genes <cit>  having 80% sequence similarity and coding for mature peptides of  <dig>  and  <dig>  kda are found as a tandem repeat in supercontig <dig> .
they are similar only to salivary peptides of ae.
albopictus <cit> .
both genes are significantly overexpressed in sialotranscriptomes.
rt-pcr suggests that these genes occur in both male and female sg.
other salivary polypeptides
additional file  <dig> lists an additional  <dig> full-length transcripts originating from  <dig> genes coding for proteins found to date only in the genus Ædes.
six of these genes have overrepresentation in the sialotranscriptome, as follows: ae- <dig> <cit> , ae- <dig> <cit> , gi| <dig> <cit> , gi| <dig> <cit> , ae- <dig> <cit> , and ae- <dig> <cit> .
rt-pcr in  <dig> of the  <dig> transcripts show enrichment or female specificity for  <dig> transcripts and ubiquitous expression for  <dig> genes, while  <dig> appear to be transcribed in both male and female sg .
their function is unknown.
proteins of possible housekeeping function
function possibly predicted
transporter function and storage proteins
being a secretory organ, mosquito sg are involved in active ion and water transport associated with their function.
v-atpases are generic 'batteries' that generate a proton gradient across membranes that can be coupled with ion exchangers and are used in eukaryotic cells for transport purposes  <cit> .
this multi-subunit enzyme complex has been extensively studied in insects, in particular in lepidotera larvæ midgut  <cit>  and in the malpighian fluid transport in mosquitoes  <cit> .
a role for v-atpases in mosquito sg secretion has been previously proposed  <cit> .
additional file  <dig> reports  <dig> gene products including  <dig> subunits of the v-atpase complex,  <dig> aquaporins ,  <dig> chloride channels, and the enzyme carbonic anhydrase, which is associated with proton transport in epithelia to speed intracellular ph regulation via the co2+h2o↔bicarbonate + h+ reaction.
probable signal transduction function
thirty-eight proteins,  <dig> of which are novel, are described in additional file  <dig> as possibly associated with signal transduction events.
included are four proteins associated with inhibition of apoptosis, four enzymes associated with juvenile hormone metabolism, one associated with ecdyesteroid metabolism, and a gamma-amino butyric  receptor-associated protein, in addition to protein kinases and phosphatases.
nuclear regulation, transcription factors, and transcription machinery
twelve  proteins are associated with nuclear function including histones, zinc finger proteins, and proteins associated with cell division.
additionally, we found  <dig> possible transcription factors and  <dig> proteins involved in the transcription machinery, only one of which has been previously reported .
ribosomal proteins and other proteins associated with the translation machinery
being an organ involved in abundant protein synthesis, it is not surprising that many ribosomal and other translation associated proteins were identified.
additional file  <dig> lists  <dig> ribosomal proteins,  <dig> of which are novel for Æ. ægypti.
twelve of these ribosomal proteins, although also found in other transcriptomes, are significantly overexpressed in the sialotranscriptome.
these are ae- <dig>  ae-198a, ae- <dig>  ae- <dig>  ae- <dig>  ae-200a, aet- <dig>  aet- <dig>  gi| <dig>  gi| <dig>  aet- <dig>  and aet- <dig>
eighteen proteins  were possibly associated with the translation machinery, including elongation factors, trna synthases, and translation initiation factors.
elongation factors  <dig>   <dig> alpha, and  <dig> beta were significantly overexpressed in the sg.
protein modification and protein export machinery
forty-four proteins  were possibly associated with the protein modification machinery including enzymes associated with proline isomerization, disulfide bridge formation, glycosylation, and several chaperones.
thirty-six proteins  are possibly associated with the protein export machinery, including signal peptidase complex, endoplasmic reticulum, golgi, and vacuole proteins.
the putative cargo transport protein emp <dig> is overexpressed in the sialotranscriptome, although also found in other nonsalivary libraries.
evidence for expression of a protein disulphide isomerase was found by ms/ms results of the tryptic digestion of band labeled pdi in figure  <dig>
oxidant metabolism
one peroxiredoxin, one thioredoxin, two superoxide dismutases, one cytochrome p- <dig> enzyme, and one truncated catalase are among ten proteins associated with oxidant metabolism .
the cytochrome p- <dig> enzyme has a signal peptide indicative of secretion and is a member of the cyp <dig> family , but it is included as a possible housekeeping function due to its high similarity to other insect enzymes <cit>  and because these enzymes normally need an associated reductase driven by nadph+, which is normally only found intracellularly.
members of the cyp <dig> family can be found in peroxisomes, where they can be associated to arachidonic acid or eicosanoid reactions  <cit> .
of interest, aet- <dig> is similar to a 40-kda farnesylated protein associated with peroxisomes <cit> , indicating the presence of this organelle in Ædes sg and the possible reason for the signal peptide that may be needed for directing this enzyme to the peroxisome.
proteasome and lysosomal machinery
seventeen proteins  were associated with the proteasome machinery, including several proteasome subunits and ubiquitin-related enzymes.
two previously described lysosomal enzymes are also listed in additional file  <dig>
cell metabolism
forty-three proteins associated with nt, aa, carbohydrate, lipid, and heme metabolism or transport are described.
aet- <dig> is similar to enzymes annotated as kynurenine formamidase  <cit>  and has a kog motif indicative of this enzyme  <cit> ;  <dig> transcripts were found among the  <dig>  salivary est but only  <dig> in the  <dig>  nonsalivary est, indicating this enzyme is overexpressed in the sg of Ædes.
kynurenine formamidase is a key enzyme in the degradation of tryptophan, producing l-kynurenine from n-formyl-kynurenine, the product of the action of l-tryptophan:oxygen  <dig> -oxidoreductase on tryptophan, and a precursor to xanthurenic acid, which has been described in the sg of an.
stephensi  <cit> .
xanthurenic acid has also been reported as the mosquito-derived gametocyte exflagellation factor of plasmodium  <cit> .
the presence of xanthurenic acid in Ædes saliva remains to be demonstrated, although a recent report indicates that Ædes mosquitoes deficient in the production of xanthurenic acid sustain normal p. gallinaceum development  <cit> .
energy metabolism
fifty-four enzymes  are presented as involved in energy metabolism.
most of these are mitochondrial constituents, a few of which are overrepresented in the salivary libraries compared with the other libraries, perhaps due to the larger-than average-salivary metabolism associated with protein synthesis and secretion.
evidence for expression of the alpha and beta subunits of the f0f1-type atp synthase were found by ms/ms data obtained from bands labeled f0f1α and f0f1β on the 2d gel  indicating their abundant expression.
cytoskeletal, adhesion, and extracellular matrix proteins
fifteen proteins  are associated with cytoskeletal, intercellular adhesion, or extracellular matrix functions, including actins, dynactin, tubulins and annexins, and the basal lamina protein named as sgs <dig>  which was found to be a sg receptor for p. gallinaceum sporozoites  <cit> .
notably, this protein family has homologues only in an.
gambiæ, where it was abundantly expressed in the sg of adult females  <cit>  and in bacteria, the most closely related protein outside anopheles being from the wolbachia  <cit> .
four such large proteins  were described in anopheles, all intronless and contained in a tandem repeat on chromosome arm 3r, while six have been identified in Ædes, also intronless <cit> , and consistent with their horizontal acquisition from a wolbachia bacteria.
for more details on this protein family, see reference  <cit> .
probably housekeeping, function unknown
fifty-nine proteins are described  that are conserved with other organisms, thus characterizing the large group of 'conserved hypothetical' proteins  <cit>  or are just hypothetical proteins with no similarities to other known proteins .
two of the conserved hypothetical proteins are clearly membrane proteins of unknown function.
several members of this group are significantly overexpressed in the sg when est's in the sialotranscriptome are compared with the remaining transcripts.
CONCLUSIONS
using high-throughput transcriptome analysis, we significantly expanded the Æ. ægypti sg transcript repertoire.
a total of  <dig> transcripts was identified,  <dig> of which are new, and mostly full length.
a subset of  <dig> transcripts was identified as possibly sg specific,  <dig> of which are novel.
analysis of tissue-specific transcription of selected genes revealed at least  <dig> genes whose expression is specific or enriched in female sg, whereas  <dig> additional genes were expressed in female sg and in males but not in other female tissues.
most of the  <dig> proteins coded by these transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents.
this catalogue makes Æ. ægypti the mosquito vector for which the most complete salivary transcriptome is available.
we hope that this updated catalogue will help our continuing effort of understanding the evolution of blood sucking in vector arthropods and the discovery of novel pharmacologically active compounds.
an unexpected finding of this work was the occurrence of four protein families specific to sg that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.
previously, the sgs family was shown to localize specifically in the basal surface of sg cells and may function as a plasmodium receptor  <cit> .
here we identify three new families of salivary and possibly secreted proteins  characterized by having uniexonic genes and psi-blast retrieval of only salivary proteins of hematophagous diptera and bacterial proteins.
although horizontal gene transfer is common in prokaryotic organisms, it is a relatively rare finding in eukaryotes  <cit> .
to the extent that these genes are really of bacterial origin, it may emphasize the unusual paths of sg gene evolution in the quest of hematophagous animals to obtain their 'perfect' potion that allows disarming of the complex host pathways of inflammation and hemostasis that would otherwise disrupt blood feeding.

 Ædes ægypti is a highly anthropophagic and cosmopolitan mosquito vector of epidemic dengue and yellow fever. to achieve fast blood feeding, adult female mosquitoes inject a complex salivary mixture into their hosts while probing for blood. mosquito saliva, like that of other blood-feeding animals, has antihemostatic and antiinflammatory activities that counteract host responses that would otherwise restrict blood flow or call the attention of the host to the feeding site. a preliminary transcriptome of adult female salivary glands has been previously reported where full-length transcripts have been described based on an analysis of expressed sequence tags. of these putative proteins, ten have been verified by edman degradation of coomassie blue-stained bands from sodium dodecyl sulfate/polyacrylamide gel electrophoresis of sg homogenates. most salivary proteins found have no known function. the genome of Æ. ægypti has been recently made available, facilitating further gene discovery. in this paper, we present the analysis of an additional set of sg cdna sequences. we describe new transcripts, of which code for proteins of a putative secretory nature, most of which have no known function. we expect this work will contribute to the understanding of the evolution of blood feeding in arthropods and to the discovery of novel pharmacologic agents.  general description of the salivary transcriptome database a total of clones was included in the est salivary database, including previously described. the average length of the sequences was bp, with sequences containing a polya signature. these clones assembled into clusters and singletons. in this paper, we will use the word'contig' to refer to clusters of one or more sequences. mitochondrial sequences, identified by their match to sequenced aedes albopictus and anopheline mitochondrial genomes, accounted for est from clusters. of interest, several of these mitochondrial sequences mapped to scaffolds named supercont and also to supercont, supercont, and supercont. upon close inspections, these genomic scaffolds contain large segments of high similarity to ae. albopictus mitochondrial genome. accordingly, these contigs may be assigned to the mitochondrial genome in the final genome assembly, or may represent translocation of ae. aegypti mitochondrial genes to the nuclear genome. to attempt a functional classification of these unique sequences, we compared them with proteome databases by blastx and with protein motifs by rpsblast. following manual annotation of these contigs, which included assignment of known or putative functions to the translation products, they were further divided into four categories: secreted with contigs and sequences; housekeeping with contigs and sequences; transposable element with contigs and sequences; and a last category composed of contigs coding for proteins of unknown function with contigs and sequences. the unknown class may contain truncated transcripts mainly mapping to 3' untranslated regions of genes. although the sclass corresponds to only 27% of the contigs, it consists of 64% of all est, reflecting the relatively low complexity and abundance of the secretory material of the sg, as indicated before. transcribed transposable elements nine transcripts in our database possibly derive from transposable elements. their translation products are similar to those of the sea urchin strongylocentrotus purpuratus and to tc1-like transposase. these transcripts may indicate active ongoing transposition activity in Æ. ægypti or, more likely, they may represent regulatory elements suppressing transposition of relatively recent genome invasions. housekeeping gene products putative hclass genes were further classified according to their possible function. results are available online and can be searched on the columns labeled "class" and "comments". more than 50% of the sequences in this class derive from transcripts associated with protein synthesis, energy metabolism, protein modification, and protein export. transporters and signal transduction gene products are also highly represented in the library. est matching transporter proteins were found for several v-type atpase subunits, na++k+ atpases, ca++ atpases, aquaporin, and several families of solute carriers. v-type atpases have been implicated in the secretion of saliva in diptera. updated catalogue of salivary proteins we used the transcriptome set and the artemis tool to identify novel proteins coded in the Æ. ægypti genome. we have also assembled our transcripts with a set of ~ est of Æ. ægypti available in public databases to obtain eventual full-length information of translation products. this assembled est dataset, similarly organized as that for anoxcel, is available online. using these tools, we identified protein sequences, mostly full length, deriving from the salivary transcriptome of adult female Æ. ægypti mosquitoes. of these protein sequences, were identified and contributed to genbank from this work, and the remaining were previously known. of these proteins, we identified a set of putative secreted proteins expressed in the sg of Æ. ægypti, of which are novel. to obtain additional information potentially useful to address future functional analysis, we determined the tissue and sex specificity of a selected subset of transcripts encoding secreted proteins and corresponding to a total of genes. these transcripts were selected based on their similarity to transcript families found in diverse mosquito species, and in the presumption that their translated products might play a role either in sugar or blood feeding. oligonucleotide primers suitable for amplification of corresponding mrna were employed for reverse transcriptase-polymerase chain reaction amplifications using as template total rna extracted from female sg, female carcasses, and whole adult males. primers amplifying the ribosomal protein s were used for normalization and as control. the results of this analysis are summarized in table we previously used a similar assay for the analysis of the anopheles gambiæ salivary transcriptome and obtained results overlapping very well with the information independently obtained by marinotti et al. using the affymetrix microarray chip. the results reported here also fit well with the data obtained comparing salivary versus nonsalivary libraries. with our assay, it is possible to distinguish three broad classes of genes. first, genes that are female sg specific or whose expression is enriched in the female glands: they are indicated as sg or enr, respectively, in table products encoded by these genes are likely to play some role in blood feeding, for example as antihemostatics or immunomodulators. among those genes analyzed, belong to this class; more precisely, were female gland specific, and were enriched in female sg. they include both genes with unknown functions and genes known from previous studies on other mosquito species to be involved in the acquisition of blood meals. a second group is represented by genes expressed in female glands and in adult males, without any expression in female carcasses: these are identified as sg,m in table it is very likely that most of these genes are gland specific and expressed both in male and female glands. the corresponding products may be involved in sugar feeding, antimicrobial activity, or other gland functions; transcripts are members of this group. finally, the last class includes genes with ubiquitous expression, i.e. expressed at approximately the same level in the three tissues examined and indicated as ubiq in table these genes most likely encode polypeptides involved in housekeeping functions: of the transcripts analyzed belong to this group. the following is a detailed description of the full-length transcripts found in the sg of adult female Æ. ægypti and of their profile of expression. the four classes are sg; enr; sg,m; and ubiq. clones marked with # were analyzed three times, all others twice. clones and may undergo alternative splicing. although clones and, and were assigned to sg class, some faint bands were seen in some samples from males. clones and underwent cycles due to low amplification levels. secreted salivary proteins proteins with some function confirmed or presumed from structure secreted ligand carrier-like proteins including d family d salivary proteins the first d7-coding gene was reported years ago, for the mosquito Æ. ægypti. it was later found in virtually all mosquito sialotranscriptomes where short and long forms were recognized. in an. gambiæ, this gene family, except for one poorly transcribed gene, appears to be selectively expressed in the female sg, indicating a role in blood feeding. this protein family is distantly related to the odorant-binding protein family, which specializes in binding small ligands. recently, some mosquito d proteins were shown to bind and inhibit the action of biogenic amines such as serotonin, histamine, and norepinephrine, a function that might help blood feeding. additionally, one short d protein from anopheles stephensi, named hamadarin, was shown to prevent kallikrein activation by factorxiia. long d forms also exist in sand flies and culicoides, indicating that this gene family was recruited very early in the evolution of hematophagous nematocera. in the mosquito an. gambiæ, five short and three long d proteins are known. their genes are organized in a inverted tandem repeat where the coding region for the three long proteins is followed by the five genes coding for the short protein in the reverse orientation. in Æ. ægypti, three short and two long d proteins map to the assembled genome supercontig, and one short d protein maps to supercontig. the genomic region coding for the d proteins in supercontig shows three short d genes followed by two genes coding for long forms; however, while in anopheles the frame orientation of the short and long forms is the same, in Ædes there is no consistent orientation. similarly to an. gambiæ, all large Ædes d genes contain five exons; however, all short Ædes d genes have two exons, including that in supercontig, while in anopheles, four of the five genes coding for short d proteins have three exons. the anopheline gene coding for the two-exon gene is poorly expressed, leading to the suggestion that this two-exon gene may be turning into a pseudogene. the differences between anopheles and Ædes in the number of d coding genes, their exon number, their orientation, and their chromosome location are consistent with the ~ million years of separation between culicines and anophelines. in agreement with the larger genome size of Ædes, the five-gene d cassette of Ædes spans nearly kb, four times more than the eight-gene cassette of anopheles. to investigate whether additional genes were expressed within the d gene cassette in Æ. ægypti, we mapped ~ est to the Æ. ægypti genome. no new identifiable expressed genes were revealed in the d region; however, in the vicinity of the short d cassette, we found two transcripts, both deriving from sg libraries, which map to the intron of d7s and to the 3' region of the same gene. translations of these two est do not reveal extended open reading frames. this finding is reminiscent to the d short gene region of anopheles, which also has an apparently noncoding est mapping to the end of the short cassette, but at its 5' end. we have hypothesized previously that these noncoding transcripts could be associated with transcriptional regulation of the cassette. alignment of the d sequences from Æ. ægypti with those of Æ. albopictus, culex quinquefasciatus, an. gambiæ, and one d salivary protein from the sand fly lutzomyia longipalpis indicates, as shown before, that the short d proteins appear to be truncated versions of the long d proteins, which appear to be the ancestral type. the resulting phylogram, using the sand fly sequence as an outgroup, shows three clades without strong bootstrap support; however, the inner tree branches show considerably more conservation of the multiple forms within genus than between genera. for example, the common long d clade shows that two of the long d proteins of an. gambiæ are more closely related to each other as are the culex or the Ædes pair. within the Ædes genus, the Æ. albopictus and Æ. ægypti homologues are distinctly grouped together, indicating that they share a relatively recent common ancestor before the duplication event, as expected from these two mosquitoes of the same subgenus. the same pattern is visible in the culicine short d clade where all short d proteins are more related to each other within genus than between genera. this is even more remarkable in the short d proteins of anopheles, where all short d proteins form a single clade outside of their culicine counterparts. if the gene duplication events that lead to the formation of long and short d proteins occurred in the primordial mosquito ancestral to both culicines and anophelines, the tree pattern observed would be one where the orthologous pairs would be more similar to each other between genera than within genus. two possible explanations may account for the observed tree pattern: either the gene duplications leading to the d expansions occurred independently after the division of the culicine and anopheline lineages, or some degree of gene conversion occurred within each species, maintaining the uniformity of the genes within species. this latter scenario is consistent with the proposed primordial role of the d proteins, e.g. sequestration of host serotonin released by thrombocytes at the site of bite, a function that would require the d proteins to be a major salivary protein constituent. the gene duplication event would be beneficial in allowing increased transcript mass needed to create the substantial amount of protein needed in the mosquito saliva to chelate the near-micromolar concentration of the vasoactive amine. gene conversion events to maintain this function on multiple genes could be beneficial at this earlier stage of blood feeding evolution. this phenomenon would maintain intraspecific copies of the gene family more similar to each other than to the orthologous interspecific copies. with time, other salivary proteins may have taken a similar role of preventing platelet function, allowing the d proteins to acquire different functions such as binding other amines or to become anti-bradykinins, a function apparently only acquired in the anophelines, which diverged from the culicines ~ million years ago. for a review on the evolution of gene families, see references. evidence for synthesis of the two large d proteins and for d7s was shown before from edman degradation results of sds-page gels. presently, we observed extensive coverage of tryptic fragments for both long d proteins as shown by two-dimensional gel electrophoresis. this protein family appears polymorphic. the predicted translation products of some of these alleles are shown in additional file as expected from this protein family, all transcripts described in additional file are more expressed in the sg cdna libraries than in the remaining libraries, three of which are significant by the χ test at the level. rt-pcr experiments agree with these results, indicating a selective or preferential expression of this gene family in female sg. it should be noted, however, that transcripts encoding the short d were exclusively found in female glands, whereas mrna encoding the long d were also detectable at a lower level in adult males and in other female tissues. this observation may be connected to an independent regulation of the short and long d cassettes, which are more that kb apart. phosphatidylethanolamine binding proteins we report three members of this very ubiquitous protein family never found before in sialotranscriptomes of blood-sucking arthropods, two of which have clear signal peptide indicative of secretion. this protein family is known to bind lipids and was also shown to have serine protease inhibitory capacity. their role in saliva is unknown. no enrichment was found for these transcripts on the sg libraries when compared with other libraries. other small molecule binding proteins an obp and a lipocalin with a juvenile hormone binding motif were found in the sialotranscriptome of Æ. ægypti, both containing distinct peptide signal indicative of secretion. the d proteins belong to the obp superfamily. lipocalins are abundantly expressed in tick and triatomine sialomes, where they act as nucleotide -and biogenic amine-binding proteins in addition to other functions. their function in Ædes is unknown. no enrichment was found for these transcripts on the sg libraries when compared with other libraries. secreted protease inhibitors serpins two serpins have been described before in Æ. ægypti, one of which has been characterized as an inhibitor of factorxa of the clotting cascade. we present one allele of the fxa-directed anticoagulant precursor having only 89% identity to the reported protein which originated from the rockefeller strain of Æ. ægypti, and two alleles of a novel salivary serpin mapping to supercontig. the three genes coding for these serpins are not located near each other in the Æ. ægypti genome. all three salivary Æ. ægypti serpins have corresponding homologues found in ae. albopictus sialotranscriptome. the novel serpin has abundant tryptic fragment matches recovered by proteomics, indicating its expression in the sg, as did gi|. transcripts for all serpins are significantly overrepresented in the sg library when compared with the remaining libraries, in accordance to the rt-pcr experiments shown in table which indicates that two of the three serpins are female specific and that one may be found also in males but not in carcasses of females not containing sg. other protease inhibitors a kazal domain-containing peptide, similar to one described in ae. albopictus and to several other proteins described as thrombin inhibitors, was found in the Æ. ægypti sialotranscriptome. a cystatin was also found, but this protein is reported as truncated; we were not successful in searching the genome for the missing exon. this could be due to the large intron size observed in ae. aegypti. accordingly, no indication of secretion is possible, but it is described in this section due to the importance of this family in inhibiting proteases associated with inflammation. both transcripts are ubiquitously found in mosquito tissues by rt-pcr and may play a housekeeping role. vasodilator sialokinin the gene coding for this endothelium-dependent peptide vasodilator has been reported earlier and shown to be transcribed specifically in female sg. although two forms of the peptide have been described earlier differing in the aminoterminal, only one gene is found coding for this peptide sequence, for which est were found in the salivary cdna library. the asn version may have been an artifact from the modification of the original peptide, which was stored in acidic solution. enzymes nucleotidases the salivary purinergic degradation machinery of Æ. ægypti comprises the enzymes apyrase, adenosine deaminase, and purine hydrolase, which may serve an antihemostatic and antiinflammatory function by removing nucleotide agonists of platelet aggregation and mast cell degranulation. in addition to these previously described enzymes, we found a second 5' nucleotidase that may function either as an alternative apyrase or as a secreted salivary 5' nucleotidase, as is the case with lutzomyia longipalpis. the novel 5' nucleotidase has only 38% identity to the previously characterized apyrase form of aedes aegypti but has a higher identity to a culex. quinquefasciatus salivary 5' -nucleotidase/apyrase protein. 5' nucleotidases are typically seen in the external part of the cellular membrane to which they are bound by a inositol phosphate anchor. secreted apyrases and 5' nucleotidases have lost either the conserved ser or the surrounding lipophylic amino acids to which the inositol phosphate moiety binds to. the novel Ædes 5' nucleotidase, like the previously described salivary apyrase, lacks the typical ser residue surrounded by hydrophobic aa typical of membrane-bound enzymes, similarly to other mosquito salivary 5' nucleotidase, supporting their role as secreted 5' nucleotidases. this novel apyrase may contribute to the purinergic degradation machinery found in saliva of Æ. ægypti. all these genes are overrepresented in sg libraries and, except for ada, significantly so. rt-pcr results are somewhat contradictory with the proposed role of these enzymes in blood feeding: with the exception of the ada coding transcript that was enriched in female sg, the other genes appeared to be sg specific, because they are expressed in sg of females and in whole males, which would suggest a role in sugar feeding, instead. we do not have a good explanation for this observation; however, we should point out that apyrase, purine nucleosidase, and ada showed very similar expression profiles by rt-pcr in the related mosquito Æ. albopictus. evidence of synthesis of these enzymes was found for the ada, the original apyrase, and the pnase, which provided abundant tryptic fragments. a novel ribonuclease of the t family was also characterized. this enzyme has not been previously characterized in sialotranscriptomes. it has a typical signal peptide indicative of secretion and may function in the degradation of extracellular rna. serine proteases nine secreted serine proteases varying in predicted mature molecular weight between and kda were found in the Æ. ægypti sialotranscriptome, seven of which are being reported for the first time. two of these serine proteases contain a cub domain, indicating specialized substrate recognition. both are found in supercontig within kb of each other. some of these enzymes are possibly related to immunity and are similar to other enzymes annotated as prophenoloxidase activators, but they could have been co-opted to function in hydrolyzing specific host proteins. a smaller number of this type of enzymes found in an. gambiæ sialotranscriptomes was selectively expressed in the sg of adult females, indicating they may play a role in blood feeding, perhaps by activating antiinflammatory pathways or deactivating inflammation. one such Ædes enzyme is ae-, similar to proteins annotated as chymotrypsin, which is overexpressed in Ædes sialotranscriptomes as opposed to the remaining transcriptomes. three of these serine proteases were significantly underrepresented in the nonsalivary-gland libraries. four serine proteases tested by rt-pcr agreed with the library frequency results: the transcripts were found in female glands and adult males but not in female carcasses deprived of sg. sugar hydrolases previously reported amylase and alpha-glycosidase/maltase are abundantly overrepresented in the salivary est collection. these genes were shown to be expressed in the proximal regions of the female glands, the region associated with sugar feeding. other hydrolases an alkaline phosphatase and a carboxylesterase, both containing signal peptides indicative of secretion, are described. both enzymatic activities in adult female sg have been previously described in Ædes, and the esterase activity shown in saliva, but their function in blood feeding can only be speculated upon. immunity-related proteins the sg of mosquitoes produce various antimicrobial polypeptides and other immunity-related products such as bacterial surface-recognizing proteins and lectins that may be important in opsonization and initiation of activation of the ppo enzyme leading to pathogen melanization. the purpose of these products may be to control microbial growth in the sugar solutions stored in the crop or in the gut following a blood meal. previously, lysozyme was found in both male and female sg of Æ. ægypti, and in the mosquito crop and shown to be secreted by females following either a sugar or blood meal. indeed, Æ. ægypti salivary lysozyme is significantly overrepresented in the sg libraries, and the rt-pcr experiment supports salivary expression in both male and female sg. anopheline mosquitoes also display a similar pattern of lysozyme expression in the proximal gland region. it is possible that most immunity-related gene products follow the same pattern of expression shown by lysozyme. some of the enzymes possibly associated with ppo activation have been listed above in the enzyme section. c-type lectins five c-type lectins are described in additional file four of which are novel. expression of ae- and gi| was confirmed by mass spectrometry following tryptic digestion of protein bands. these two lectins are also expressed significantly more in sialotranscriptomes than in other Æ. ægypti cdna libraries, indicating that they are possibly salivary-tissue specific. in accordance with this expectation, rt-pcr experiments indicate expression exclusively in female sg. the two genes coding for these salivary-specific lectins were found as an inverted tandem repeat in supercontig, each with a single intron separating the signal peptide gene region from the remaining coding sequence. two other c-type lectins tested by rt-pcr were ubiquitously expressed. the c-type lectin family is expressed in most mosquito sialotranscriptomes described thus far. this protein family is implicated in immune recognition phenomena in general and in plasmodium development in anopheles in particular. despite these not-yet-demonstrated roles of c-type lectins in salivary immunity, it is interesting that in snake venoms this protein family has been recruited to perform various unrelated functions such as anticlotting, toxin, and platelet aggregation inducer. lectins may also play a role in the colectin pathway of complement activation. hemagglutinins were described in anopheline sg more than years ago. this activity may help concentration of red blood cells in the mosquito gut. the molecular nature of any anopheline hemagglutinin, however, is unknown. differently from anophelines, and despite having salivary lectins, Ædes sg homogenates lack hemagglutinins, indicating that the salivary lectins do not recognize vertebrate red blood cells or that they are monomeric in their carbohydrate binding site. overall, it appears that the two female sg-specific lectins may have a role in hemostasis rather than immunity. other proteins with sugar-binding domains aet- and aet- are similar to, but shorter than, n-acetylgalactosaminyltransferase and glucuronyltransferase, respectively, appearing to derive from novel genes that arose from gene duplications and partial deletions of ancestral genes coding for carbohydrate binding enzymes, the final products lacking the original carboxyterminal domain. aet- has a partial pfam glycos_transf_ motif that comprises a diverse family transferring sugar from udp-glucose, udp-n-acetyl-galactosamine, gdp-mannose, or cdp-abequose to a range of substrates including cellulose, dolichol phosphate, and teichoic acids. aet- has a weak match to the pfam udpgt motif and is similar to proteins in the nonredundant database annotated as udp-glucosyl transferase. it is possible that these proteins have a destination in the endoplasmic reticulum or golgi and do not have a secretory nature. their function is unknown. the proteins annotated as imaginal disk growth factor protein and aea-871bre were expressed in the sialotranscriptome of Æ. ægypti. these proteins have a chitinase domain and are homologous to an. gambiæ bacteria responsive protein and bacteria responsive protein, which were shown to be immune-responsive chitinase-like proteins that have lost chitin-binding activity. angiopoietins/ficolins this group of proteins has the pfam fibrinogen c motif seen in invertebrate proteins displaying lectin activity toward n-acetylglucosamine residues and implicated in immune function. in an. gambiæ, the ficolin family was expanded in comparison to drosophila melanogaster, where members were seen in its genome as opposed to in the fruit fly. three proteins belonging to this family are shown in additional file two of which are novel. evidence for salivary expression of gi| and ae- was found by mass spectrometry in tryptic digests of protein bands. of interest, the two genes for these proteins occur as a tandem repeat in supercontig. est for these two genes are also overrepresented in the sialotranscriptomes and are indicated to be female-salivary-gland specific by rt-pcr experiments, thus suggesting a blood-feeding rather than an immune role for these proteins. antimicrobial peptides the gene products for the amp gambicin, lysozyme, and defensin a have been previously described in Æ. ægypti sialotranscriptomes and are listed in additional file transcripts encoding gambicin and defensin a were detected by rt-pcr in all tissues examined, indicating ubiquitous expression; however, a significant overrepresentation of the corresponding est in sialotranscriptomes should be pointed out. we additionally describe three novel peptides that may have an antimicrobial function. aet- has gy repeats that are also found in peptides of similar size known to have antimicrobial activity in nematodes. aet- and aet- are candidate amp containing a hhh motif seen in other histidine-rich amp. aet- appears to be sg specific, as a total of est was found in the combined sg transcriptome, although none were seen in other tissue transcriptomes. rt-pcr confirmed the presence of the transcript in female glands and male bodies but not in female carcasses without sg. other immune-related gene products a peptide closely related to a previously described Æ. ægypti peptide named i23r potentially involved in plasmodium susceptibility was found in the sialotranscriptome. we also present an allele to aea-233a, indicating the polymorphism of this gene. the Æ. albopictus sialotranscriptome revealed a homologue that is 63% identical, but no significant matches were found to any other animal or plant proteins in the nr database. this peptide may belong to a not-yet-characterized antimicrobial gene family specific to the Ædes genus. expression of aea-233a was ubiquitous by rt-pcr. two other gene products are described, both associated with pathogen surface-pattern recognition: the previously described gram negative binding protein, which is significantly overrepresented in sialotranscriptomes and appears expressed both in female sg and in adult males, and the novel ae-, which is similar to peptidoglycan recognition proteins and was ubiquitously expressed by rt-pcr experiments. mucins mucins and peritrophins are proteins associated with lining of epithelia or inert extracellular structures, such as chitin. mucins are highly glycosylated proteins containing ser or thr modified with n-acetylgalactosamine residues. their expression in the sg may have a function of lining the chitin surfaces of the mouthparts, but they may also assist in antimicrobial functions. we present mucins in additional file of which are novel, including one allele. these proteins have an average ser+thr equal to % of their total aa, as opposed to % observed as the average of all proteins found in additional file we additionally report on a polypeptide containing three glycosylation sites and one chitin-binding domain, which may be involved in proximal lining of the cuticular duct. all other proteins have 11– glycosylation sites. putative secreted proteins without functional classification belonging to ubiquitous protein families antigen family ag5-related salivary products are members of a group of secreted proteins that belong to the cap family. members of this protein family are found in the sg of many blood-sucking insects. most of these animal proteins have no known function; in the few instances to the contrary, they diverge from proteolytic activity in conus, to smooth muscle-relaxing activity in snake venoms, to salivary neurotoxin in the venomous lizard heloderma horridum. three members of this gene family were previously described in the sialotranscriptome of Æ. ægypti. est's for all three genes are overrepresented in the sialotranscriptome as compared with the combined transcriptomes, indicating they may be preferentially expressed in the sg. in accordance with these results, gi| was exclusively transcribed in female glands as indicated by rt-pcr, suggesting an antihemostatic function for the gene product, while the other two genes are transcribed in female glands and male bodies but not in female carcasses. differently to an. gambiæ, which has four salivary ag members, three of which cluster in chromosome arm l, the salivary ag proteins of Ædes do not appear to cluster in the genome, mapping to different supercontigs. other secreted proteins of unknown function found in non-bloodsucking insects eight putative secreted proteins have similarities to proteins or protein domains found in non-bloodsucking insects. one of these proteins is a truncated fragment where it is not possible to identify whether it has a signal peptide indicative of secretion, but it has a weak cdd domain pan_ap_hgf, which is found in plasminogen/hepatocyte growth factor proteins, and various proteins found in bilateria, such as leech antiplatelet proteins; however, the mrna encoding this protein was found ubiquitously expressed by rt-pcr and may not have a unique salivary role in blood feeding. ae- has a til domain and is significantly overexpressed in sialotranscriptomes. rt-pcr indicates both male and female sg may be the target tissue of expression of this peptide. peptides containing a til domain were also found in the an. stephensi and an. gambiae adult male sialotranscriptomes. the finding of this type of peptide being overexpressed in male an. gambiæ sg indicated a possible antimicrobial function rather than a function as a host serine protease inhibitor during blood feeding. indeed, a tick til domain containing peptide named ixodidin was found to have an antimicrobial function in addition to inhibiting serine proteases. the remaining six polypeptides have similarities to drosophila or other species, and their structure does not hint at any particular function. belonging to families only found in blood-sucking diptera 56-kda family this protein family has been found to date only in salivary transcriptomes of adult mosquitoes, including adult male an. gambiæ. the sg specificity of this gene transcript in Æ. ægypti is supported by significant overrepresentation of est on the sialotranscriptome and by rt-pcr. all family members have a signal peptide indicative of secretion and a predicted molecular weight near kda. blast comparisons also show weak similarity to bacterial proteins but to no other eukaryotes. following iterations of psi-blast, only mosquito and bacterial proteins are retrieved, suggesting that this family of proteins may have originated as a lateral transfer from a bacterial genome to the ancestral mosquito genome. the single exon structure of the gene – unusual in eukaryotes, particularly for a protein of this size, but the rule in prokaryotes – supports this hypothesis. the an. gambiæ homologue also displays a single exon gene structure, as reported previously. the bacterial proteins retrieved by psi-blast are mostly annotated as phage-associated proteins, suggesting the lateral transfer might have occurred via a phage-associated mechanism. 41-kda family two novel alleles coding for this protein family are described in additional file the gene coding for this protein has a exon structure on supercontig. the gene product shows similarities to proteins of comparable size found in Æ. albopictus and culex pipiens and to a shorter protein in salivary transcriptomes of culicoides sonorensis. a match to Æ. ægypti gi| probably represents a misannotated protein with a dna frame-shift error. three iterations of psi-blast only retrieved culicine and culicoides proteins, indicating the uniqueness of this protein family. no salivary anopheline sequences similar to the 41-kda family have been reported, including all ensembl-predicted an. gambiæ proteins deposited at the national center for biotechnology information. est coding for the two alleles in Æ. ægypti are significantly underrepresented in the non-sg libraries; rt-pcr suggests ubiquitous expression of one of the alleles, although only very faint bands were detected even after cycles of amplification. the function of any member of this protein family is unknown. 30-kda ge-rich family this acidic, gly/glu-rich protein family is abundantly expressed in adult female mosquito sg, where they appear to be involved in allergic reactions to mosquito bites. in Æ. ægypti, two proteins of this family have been previously reported, and we now report two additional splice variants and alleles. evidence for expression was found in bands labeled ag in the 2d gel experiment shown in figure the two proteins are coded by an inverted tandem repeat in supercontig separated by only base pairs. the sialotranscriptome is significantly overrepresented in est coding for this protein family, indicating it is salivary specific. rt-pcr confirms the female sg specificity of these transcripts. based on the public sequences available, it appears that in anopheline mosquitoes, only one polymorphic gene exists for this protein family per genome. 29-kda family two different transcripts in Æ. ægypti are possibly obtained from the same genomic region coding for the basic salivary protein ae- and for the alternative shorter transcript ae-236a, which was not found on the salivary est but rather as one contig assembled from four est deriving from nonsalivary libraries. blast comparison of the deducted salivary protein with the nr database shows similarities to other culicine and anopheline salivary proteins, including weak similarities to some members of the kda protein family. four iterations of psi-blast are able to assemble only salivary proteins of mosquitoes, culicoides, and phlebotomus, including all 30-kda proteins discussed above, suggesting that either this unique protein family was co-opted as salivary proteins independently by these different families of diptera or that they have a common blood-feeding ancestor. clustalw alignment of the sequences shows that following the signal peptide region, a subset of the proteins have a ser/thr/gly-rich region, poor in aliphatic aa as shown in figure by the richness in brown residues. the carboxyterminal region is marked by the alternation of polar and aliphatic residues. a subset of culicoides proteins does not have this domain. the phylogenetic tree shows a robust mosquito clade with three members of the family per Ædes species and one member per anopheline species, which have only a single 30-kda member. a single cx. pipiens sequence is also part of this clade. cladeii has two very similar culicoides proteins. cladeiii has very divergent culex and phlebotomus sequences, and cladeiv has solely culicoides sequences, representing those lacking the ser/thr/gly-rich aminoterminal domain. it is tempting to speculate that these data support a common origin of blood feeding for these three dipteran families, where two genes are found in culicines, a single in anophelines and possibly phlebotomus, and a rather large gene expansion in culicoides, which has at least seven genes in the family. notice that cladeiii, containing mosquito and sand fly sequences, roots with culicoides cladeiv and that culicoides cladeii roots with the mosquito cladei, indicating that culicoides may have shared a common ancestor with mosquitoes that had two genes of this unique family. alternatively, convergent evolution may have shaped these genes to produce similar proteins. ae-236a was enriched in the sg of adult females, while ae- was significantly underrepresented in non-sg libraries, suggesting a salivary specificity for this protein family, as is the case with the related 30-kda family discussed above. other mosquito-or diptera-specific peptides additional file also includes additional polypeptides, one of which is an allele, showing sequence similarities to putative proteins from other hematophagous diptera including, in a few cases, some weak similarities to drosophila; of these are novel. among this class of polypeptides, nine were analyzed by rt-pcr, and seven are significantly underrepresented in the non-sialotranscriptomes, as follows: ae-, which is similar to drosophila and culicoides proteins of unknown function and was ubiquitously expressed by rt-pcr experiments; two alleles coding for basic 29-kda proteins containing putative galactosylated ser/thr and similar to culex and culicoides salivary proteins; three polypeptides whose expression appeared gland specific, as suggested by rt-pcr. ae-, is weakly similar to proteins varying in size from 150–180aa residues from anopheles and drosophila. two additional transcripts, ae- and ae- were expressed solely in the adult female sg by rt-pcr. the function of these proteins remains to be investigated. genes belonging to protein families found to date only in Ædes genus nineteen genes were found expressed in the sialotranscriptome of Æ. ægypti coding for polypeptide families known only in the Ædes genus, as follows: 62-kda family two single exon genes separated by ~ kb in supercontig code for proteins with signal peptides and mature mass of 62– kda. transcripts for these genes are significantly overrepresented in the sialotranscriptome and are shown to be adult female sg specific by rt-pcr. they are similar to homologous salivary protein sequences seen in ae. albopictus and, to a much smaller degree, to rhoptry proteins of plasmodium. repeated leu and glu residues provide similarities to myosin, indicating this protein family may be involved in adhesion phenomena. their uniqueness among metazoan and single-exon structure indicates possible horizontal acquisition of this gene family in Ædes. both genes are abundantly expressed in the sg as evidenced by bands labeled k by 2d gel electrophoresis ms/ms. 34-kda gene family seven transcripts coding for related proteins were found mapping to supercontig. after locating the corresponding genomic regions, these transcripts were annotated as truncated forms or alleles of three genes found as a tandem repeat. we additionally found one possible related gene in the most distal region of the 34-kda cassette. except for the first gene on the cassette, which codes for a 16-kda protein and has two exons, the remaining genes are single exonic and code for proteins of ~ kda. the two central genes, each with a single exon, are abundantly expressed as evidenced by ms/ms sequencing of tryptic digested bands. all transcripts matching this gene region are significantly overexpressed in the sg transcriptome when compared with the remaining libraries. rt-pcr indicates they are enriched or exclusive of adult female sg. protein products of these genes match significantly only ae. albopictus proteins. psi-blast for each transcript against the nr protein database reveals cytoskeletal proteins such as actin and myosin, mainly due to the presence of repeated charged aa. this indicates that this protein family may be associated with adhesion phenomena. the single-exon nature of most members of this gene family and their uniqueness among metazoa points to a horizontal acquisition of this gene. -kda family two genes coding for proteins of ~ -kda are found as a tandem repeat on supercontig. the gene coding for gi| is abundantly expressed as evidenced by ms/ms of tryptic digested band labeled in figure these proteins are similar only to homologues found in ae. albopictus. both genes are significantly overtranscribed in the sialotranscriptome when compared with other transcriptomes. rt-pcr indicates enrichment in the female sg or exclusive expression in the same organ. 9-kda family two genes having 80% sequence similarity and coding for mature peptides of and kda are found as a tandem repeat in supercontig. they are similar only to salivary peptides of ae. albopictus. both genes are significantly overexpressed in sialotranscriptomes. rt-pcr suggests that these genes occur in both male and female sg. other salivary polypeptides additional file lists an additional full-length transcripts originating from genes coding for proteins found to date only in the genus Ædes. six of these genes have overrepresentation in the sialotranscriptome, as follows: ae-, ae-, gi|, gi|, ae-, and ae-. rt-pcr in of the transcripts show enrichment or female specificity for transcripts and ubiquitous expression for genes, while appear to be transcribed in both male and female sg. their function is unknown. proteins of possible housekeeping function function possibly predicted transporter function and storage proteins being a secretory organ, mosquito sg are involved in active ion and water transport associated with their function. v-atpases are generic'batteries' that generate a proton gradient across membranes that can be coupled with ion exchangers and are used in eukaryotic cells for transport purposes. this multi-subunit enzyme complex has been extensively studied in insects, in particular in lepidotera larvæ midgut and in the malpighian fluid transport in mosquitoes. a role for v-atpases in mosquito sg secretion has been previously proposed. additional file reports gene products including subunits of the v-atpase complex, aquaporins, chloride channels, and the enzyme carbonic anhydrase, which is associated with proton transport in epithelia to speed intracellular ph regulation via the co2+h2o↔bicarbonate + h+ reaction. probable signal transduction function thirty-eight proteins, of which are novel, are described in additional file as possibly associated with signal transduction events. included are four proteins associated with inhibition of apoptosis, four enzymes associated with juvenile hormone metabolism, one associated with ecdyesteroid metabolism, and a gamma-amino butyric receptor-associated protein, in addition to protein kinases and phosphatases. nuclear regulation, transcription factors, and transcription machinery twelve proteins are associated with nuclear function including histones, zinc finger proteins, and proteins associated with cell division. additionally, we found possible transcription factors and proteins involved in the transcription machinery, only one of which has been previously reported. ribosomal proteins and other proteins associated with the translation machinery being an organ involved in abundant protein synthesis, it is not surprising that many ribosomal and other translation associated proteins were identified. additional file lists ribosomal proteins, of which are novel for Æ. ægypti. twelve of these ribosomal proteins, although also found in other transcriptomes, are significantly overexpressed in the sialotranscriptome. these are ae- ae-198a, ae- ae- ae- ae-200a, aet- aet- gi| gi| aet- and aet- eighteen proteins were possibly associated with the translation machinery, including elongation factors, trna synthases, and translation initiation factors. elongation factors alpha, and beta were significantly overexpressed in the sg. protein modification and protein export machinery forty-four proteins were possibly associated with the protein modification machinery including enzymes associated with proline isomerization, disulfide bridge formation, glycosylation, and several chaperones. thirty-six proteins are possibly associated with the protein export machinery, including signal peptidase complex, endoplasmic reticulum, golgi, and vacuole proteins. the putative cargo transport protein emp is overexpressed in the sialotranscriptome, although also found in other nonsalivary libraries. evidence for expression of a protein disulphide isomerase was found by ms/ms results of the tryptic digestion of band labeled pdi in figure oxidant metabolism one peroxiredoxin, one thioredoxin, two superoxide dismutases, one cytochrome p- enzyme, and one truncated catalase are among ten proteins associated with oxidant metabolism. the cytochrome p- enzyme has a signal peptide indicative of secretion and is a member of the cyp family, but it is included as a possible housekeeping function due to its high similarity to other insect enzymes and because these enzymes normally need an associated reductase driven by nadph+, which is normally only found intracellularly. members of the cyp family can be found in peroxisomes, where they can be associated to arachidonic acid or eicosanoid reactions. of interest, aet- is similar to a 40-kda farnesylated protein associated with peroxisomes, indicating the presence of this organelle in Ædes sg and the possible reason for the signal peptide that may be needed for directing this enzyme to the peroxisome. proteasome and lysosomal machinery seventeen proteins were associated with the proteasome machinery, including several proteasome subunits and ubiquitin-related enzymes. two previously described lysosomal enzymes are also listed in additional file cell metabolism forty-three proteins associated with nt, aa, carbohydrate, lipid, and heme metabolism or transport are described. aet- is similar to enzymes annotated as kynurenine formamidase and has a kog motif indicative of this enzyme; transcripts were found among the salivary est but only in the nonsalivary est, indicating this enzyme is overexpressed in the sg of Ædes. kynurenine formamidase is a key enzyme in the degradation of tryptophan, producing l-kynurenine from n-formyl-kynurenine, the product of the action of l-tryptophan:oxygen -oxidoreductase on tryptophan, and a precursor to xanthurenic acid, which has been described in the sg of an. stephensi. xanthurenic acid has also been reported as the mosquito-derived gametocyte exflagellation factor of plasmodium. the presence of xanthurenic acid in Ædes saliva remains to be demonstrated, although a recent report indicates that Ædes mosquitoes deficient in the production of xanthurenic acid sustain normal p. gallinaceum development. energy metabolism fifty-four enzymes are presented as involved in energy metabolism. most of these are mitochondrial constituents, a few of which are overrepresented in the salivary libraries compared with the other libraries, perhaps due to the larger-than average-salivary metabolism associated with protein synthesis and secretion. evidence for expression of the alpha and beta subunits of the f0f1-type atp synthase were found by ms/ms data obtained from bands labeled f0f1α and f0f1β on the 2d gel indicating their abundant expression. cytoskeletal, adhesion, and extracellular matrix proteins fifteen proteins are associated with cytoskeletal, intercellular adhesion, or extracellular matrix functions, including actins, dynactin, tubulins and annexins, and the basal lamina protein named as sgs which was found to be a sg receptor for p. gallinaceum sporozoites. notably, this protein family has homologues only in an. gambiæ, where it was abundantly expressed in the sg of adult females and in bacteria, the most closely related protein outside anopheles being from the wolbachia. four such large proteins were described in anopheles, all intronless and contained in a tandem repeat on chromosome arm 3r, while six have been identified in Ædes, also intronless, and consistent with their horizontal acquisition from a wolbachia bacteria. for more details on this protein family, see reference. probably housekeeping, function unknown fifty-nine proteins are described that are conserved with other organisms, thus characterizing the large group of'conserved hypothetical' proteins or are just hypothetical proteins with no similarities to other known proteins. two of the conserved hypothetical proteins are clearly membrane proteins of unknown function. several members of this group are significantly overexpressed in the sg when est's in the sialotranscriptome are compared with the remaining transcripts.  using high-throughput transcriptome analysis, we significantly expanded the Æ. ægypti sg transcript repertoire. a total of transcripts was identified, of which are new, and mostly full length. a subset of transcripts was identified as possibly sg specific, of which are novel. analysis of tissue-specific transcription of selected genes revealed at least genes whose expression is specific or enriched in female sg, whereas additional genes were expressed in female sg and in males but not in other female tissues. most of the proteins coded by these transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. this catalogue makes Æ. ægypti the mosquito vector for which the most complete salivary transcriptome is available. we hope that this updated catalogue will help our continuing effort of understanding the evolution of blood sucking in vector arthropods and the discovery of novel pharmacologically active compounds. an unexpected finding of this work was the occurrence of four protein families specific to sg that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes. previously, the sgs family was shown to localize specifically in the basal surface of sg cells and may function as a plasmodium receptor. here we identify three new families of salivary and possibly secreted proteins characterized by having uniexonic genes and psi-blast retrieval of only salivary proteins of hematophagous diptera and bacterial proteins. although horizontal gene transfer is common in prokaryotic organisms, it is a relatively rare finding in eukaryotes. to the extent that these genes are really of bacterial origin, it may emphasize the unusual paths of sg gene evolution in the quest of hematophagous animals to obtain their'perfect' potion that allows disarming of the complex host pathways of inflammation and hemostasis that would otherwise disrupt blood feeding. 
overall, this paper contributes to the novel identification of new transcripts, or near 3% of the Æ. ægypti proteome assuming a -protein set, and to the best-described sialome of any blood-feeding insect. most of the proteins coded by these sg transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. to expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of expressed sequence tags from cdna libraries of adult female mosquitoes. a nonredundant catalogue of transcripts is described, including coding for proteins of a putative secretory nature. additionally, a two-dimensional gel electrophoresis of salivary gland homogenates followed by tryptic digestion of selected protein bands and ms/ms analysis revealed the expression of proteins. an unexpected finding is the occurrence of four protein families specific to sg that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes. analysis of tissue-specific transcription of a subset of these genes revealed at least genes whose expression is specific or enriched in female sg, whereas additional genes were expressed in female sg and in males but not in other female tissues.
 saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. to expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of expressed sequence tags from cdna libraries of adult female mosquitoes.  a nonredundant catalogue of transcripts is described, including coding for proteins of a putative secretory nature. additionally, a two-dimensional gel electrophoresis of salivary gland homogenates followed by tryptic digestion of selected protein bands and ms/ms analysis revealed the expression of proteins. analysis of tissue-specific transcription of a subset of these genes revealed at least genes whose expression is specific or enriched in female sg, whereas additional genes were expressed in female sg and in males but not in other female tissues. most of the proteins coded by these sg transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. an unexpected finding is the occurrence of four protein families specific to sg that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.  overall, this paper contributes to the novel identification of new transcripts, or near 3% of the Æ. ægypti proteome assuming a -protein set, and to the best-described sialome of any blood-feeding insect. 