The first D7-coding gene was reported 15 years ago, for the mosquito Æ. ægypti 14. It was later found in virtually all mosquito sialotranscriptomes where short (~15 kDa) and long (~30 kDa) forms were recognized 15. In An. gambiæ, this gene family, except for one poorly transcribed gene, appears to be selectively expressed in the female SG 1718, indicating a role in blood feeding. This protein family is distantly related to the odorant-binding protein (OBP) family, which specializes in binding small ligands 17. Recently, some mosquito D7 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 18. Additionally, one short D7 protein from Anopheles stephensi, named hamadarin, was shown to prevent kallikrein activation by FactorXIIa 19. Long D7 forms also exist in sand flies 16 and Culicoides 20, 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 D7 proteins are known. Their genes are organized in a inverted tandem repeat21 where the coding region for the three long proteins is followed by the five genes coding for the short protein in the reverse orientation 12. In Æ. ægypti, three short and two long D7 proteins map to the assembled genome supercontig1.204 (Figure 1), and one short D7 protein maps to supercontig1.253 (not shown). The genomic region coding for the D7 proteins in supercontig1.204 shows three short D7 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 (but the short and long forms are in reverse orientation to each other), in Ædes there is no consistent orientation (Figure 1A). Similarly to An. gambiæ, all large Ædes D7 genes contain five exons (Figure 1B); however, all short Ædes D7 genes have two exons (Figure 1C), including that in supercontig1.253 (not shown), while in Anopheles, four of the five genes coding for short D7 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 18. The differences between Anopheles and Ædes in the number of D7 coding genes, their exon number, their orientation, and their chromosome location (in Anopheles, all eight genes are clustered, while one Ædes gene is far apart from the main cluster of five genes) are consistent with the ~150 million years of separation between culicines and anophelines 22.

In agreement with the larger genome size of Ædes 23, the five-gene D7 cassette of Ædes spans nearly 80 kb, four times more than the eight-gene cassette of Anopheles. To investigate whether additional genes were expressed within the D7 gene cassette in Æ. ægypti, we mapped ~220,000 EST to the Æ. ægypti genome. No new identifiable expressed genes were revealed in the D7 region; however, in the vicinity of the short D7 cassette, we found two transcripts, both deriving from SG libraries, which map to the intron of D7s3 and to the 3' region of the same gene (Figure 2). Translations of these two EST do not reveal extended open reading frames. This finding is reminiscent to the D7 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 D7 sequences from Æ. ægypti with those of Æ. albopictus, Culex quinquefasciatus, An. gambiæ, and one D7 salivary protein from the sand fly Lutzomyia longipalpis indicates, as shown before, that the short D7 proteins appear to be truncated versions of the long D7 proteins, which appear to be the ancestral type (Figure 3A). 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 D7 clade (Figure 3B) shows that two of the long D7 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 D7 clade (Figure 3B) where all short D7 proteins are more related to each other within genus than between genera. This is even more remarkable in the short D7 proteins of Anopheles, where all short D7 proteins form a single clade outside of their culicine counterparts. If the gene duplication events that lead to the formation of long and short D7 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 D7 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 D7 proteins, e.g. sequestration of host serotonin released by thrombocytes at the site of bite, a function that would require the D7 proteins to be a major salivary protein constituent 3. 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 D7 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 ~150 million years ago 22. For a review on the evolution of gene families, see references 2425.

Evidence for synthesis of the two large D7 proteins and for D7s2 was shown before from Edman degradation results of SDS-PAGE gels 3. Presently, we observed extensive coverage of tryptic fragments for both long D7 proteins (D7l1 and D7l2) as shown by two-dimensional (2D) gel electrophoresis (Figure 4 and Additional File 2) 14. This protein family appears polymorphic. The predicted translation products of some of these alleles are shown in Additional File 2. As expected from this protein family, all transcripts described in Additional File 2 are more expressed in the SG cDNA libraries than in the remaining libraries, three of which are significant by the χ² test at the 0.05 level. RT-PCR experiments agree with these results, indicating a selective or preferential expression of this gene family in female SG (Figure 5 and Table 2). It should be noted, however, that transcripts encoding the short D7 were exclusively found in female glands, whereas mRNA encoding the long D7 were also detectable at a lower level in adult males (D7l1, D7l2) and in other female tissues (D7l2). This observation may be connected to an independent regulation of the short and long D7 cassettes, which are more that 30 kb apart.

We report three members (one possibly truncated) of this very ubiquitous protein family never found before in sialotranscriptomes of blood-sucking arthropods 2627, 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.

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 D7 proteins belong to the OBP superfamily. Lipocalins are abundantly expressed in tick and triatomine sialomes, where they act as nucleotide (nt)-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.

Two serpins have been described before in Æ. ægypti, one of which has been characterized as an inhibitor of FactorXa of the clotting cascade 2829. We present one allele of the FXa-directed anticoagulant precursor having only 89% identity to the reported protein30 which originated from the Rockefeller strain of Æ. ægypti, and two alleles of a novel salivary serpin mapping to supercontig1.65. 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 sialotranscriptome31. The novel serpin has abundant tryptic fragment matches recovered by proteomics (band marked Serp2, Figure 4), indicating its expression in the SG, as did gi|18568304 (marked Serp1, Figure 4). 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 2, 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 (Figure 5).

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(s). 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.

The gene coding for this endothelium-dependent peptide vasodilator 3233 has been reported earlier and shown to be transcribed specifically in female SG 34. Although two forms of the peptide have been described earlier differing in the aminoterminal (aspartate or asparagine), only one gene is found coding for this peptide sequence, for which 60 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.

The salivary purinergic degradation machinery of Æ. ægypti comprises the enzymes apyrase (a member of the 5' nucleotidase family), adenosine deaminase (ADA), and purine hydrolase 353637, 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 38. The novel 5' nucleotidase has only 38% identity to the previously characterized apyrase form of Aedes aegypti39 but has a higher identity (52%) to a Culex. quinquefasciatus salivary 5' -nucleotidase/apyrase protein40. 5' nucleotidases are typically seen in the external part of the cellular membrane to which they are bound by a inositol phosphate anchor 414243. Secreted apyrases and 5' nucleotidases have lost either the conserved Ser or the surrounding lipophylic amino acids (aa) (or both) to which the inositol phosphate moiety binds to 3538. The novel Ædes 5' nucleotidase, like the previously described salivary apyrase 35, lacks the typical Ser residue surrounded by hydrophobic aa typical of membrane-bound enzymes, similarly to other mosquito salivary 5' nucleotidase (Figure 6), 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 (PNase), and ADA showed very similar expression profiles by RT-PCR in the related mosquito Æ. albopictus (Arcà et al., manuscript in preparation). Evidence of synthesis of these enzymes was found for the ADA, the original apyrase, and the PNase, which provided abundant tryptic fragments (Figure 4, bands labeled ADA, apyrase, and PNase).

A novel ribonuclease of the T2 family44 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 45.

Nine secreted serine proteases varying in predicted mature molecular weight between 28 and 43 kDa were found in the Æ. ægypti sialotranscriptome, seven of which are being reported for the first time (Additional File 2). Two of these serine proteases (AEA-87646and AEA-56247) contain a CUB domain48, indicating specialized substrate recognition. Both are found in supercontig1.217 within 63 kb of each other. Some of these enzymes (such as gi|1856833449) are possibly related to immunity and are similar to other enzymes annotated as prophenoloxidase (PPO)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 (such as protein C) or deactivating inflammation. One such Ædes enzyme is AE-22650, 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.

Previously reported amylase5152 and alpha-glycosidase/maltase5354 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.

An alkaline phosphatase55 and a carboxylesterase56, both containing signal peptides indicative of secretion, are described. Both enzymatic activities in adult female SG have been previously described in Ædes 57, and the esterase activity shown in saliva, but their function in blood feeding can only be speculated upon.

Five C-type lectins are described in Additional File 2, four of which are novel. Expression of AE-189 and gi|18568318 was confirmed by mass spectrometry following tryptic digestion of protein bands (labeled C-Lec1 and C-Lec2 in Figure 4). 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 (Table 2 and Figure 5). The two genes coding for these salivary-specific lectins were found as an inverted tandem repeat in supercontig1.10, 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 6263. 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 6465. Lectins may also play a role in the colectin pathway of complement activation 66. Hemagglutinins were described in anopheline SG more than 60 years ago 67. This activity may help concentration of red blood cells in the mosquito gut 68. 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.

AET-12005 and AET-670 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-12005 has a partial Pfam Glycos_transf_2 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-670 has a weak match to the PFAM UDPGT motif and is similar to proteins in the nonredundant (NR) 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 469 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 170 and bacteria responsive protein 271, which were shown to be immune-responsive chitinase-like proteins that have lost chitin-binding activity 72.

This group of proteins has the PFAM fibrinogen C motif73seen in invertebrate proteins displaying lectin activity toward N-acetylglucosamine residues and implicated in immune function 74. In An. gambiæ, the ficolin family was expanded in comparison to Drosophila melanogaster, where 53 members were seen in its genome as opposed to 20 in the fruit fly 75. Three proteins belonging to this family are shown in Additional File 2, two of which are novel. Evidence for salivary expression of gi|18568298 and AE-154 was found by mass spectrometry in tryptic digests of protein bands (labeled Ang1 and Ang2, respectively, in Figure 4). Of interest, the two genes for these proteins occur as a tandem repeat in supercontig1.15. 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.

The gene products for the AMP gambicin76, lysozyme58, and defensin A177 have been previously described in Æ. ægypti sialotranscriptomes and are listed in Additional File 2. Transcripts encoding gambicin and defensin A1 were detected by RT-PCR in all tissues examined, indicating ubiquitous expression (Table 2 and Figure 5); 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-59078 has GY repeats that are also found in peptides of similar size known to have antimicrobial activity in nematodes 79. AET-46280 and AET-1135881 are candidate AMP containing a HHH motif seen in other histidine-rich AMP 8283. AET-11358 appears to be SG specific, as a total of 88 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 (Table 2 and Figure 5).

A peptide (named AEA-23384) closely related to a previously described Æ. ægypti peptide named i23R85 potentially involved in Plasmodium susceptibility 86 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 protein87, which is significantly overrepresented in sialotranscriptomes and appears expressed both in female SG and in adult males (Table 2, Figure 5), and the novel AE-721088, which is similar to peptidoglycan recognition proteins and was ubiquitously expressed by RT-PCR experiments.

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 12 mucins in Additional File 2, 11 of which are novel, including one allele. These proteins have an average Ser+Thr equal to 13.8% of their total aa, as opposed to 0.9% observed as the average of all proteins found in Additional File 2. We additionally report on a polypeptide (AE-466, mucin-like peritrophin) containing three glycosylation sites and one chitin-binding domain, which may be involved in proximal lining of the cuticular duct. All other proteins have 11–69 glycosylation sites.

AG5-related salivary products are members of a group of secreted proteins that belong to the CAP family (cysteine-rich secretory proteins; AG5 proteins of insects; pathogenesis-related protein 1 of plants) 89. Members of this protein family are found in the SG of many blood-sucking insects 39091. Most of these animal proteins have no known function; in the few instances to the contrary, they diverge from proteolytic activity in Conus 92, to smooth muscle-relaxing activity 9394 in snake venoms, to salivary neurotoxin in the venomous lizard Heloderma horridum 95. 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|18568284 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 AG5 members, three of which cluster in chromosome arm 2 L 12, the salivary AG5 proteins of Ædes do not appear to cluster in the genome, mapping to different supercontigs.

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 (Figure 5 and Table 2). All family members have a signal peptide indicative of secretion and a predicted molecular weight near 56 kDa. BLAST comparisons106 also show weak similarity to bacterial proteins but to no other eukaryotes. Following 4 iterations of PSI-BLAST107, only mosquito and bacterial proteins are retrieved 108, 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 gene109 – 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 110, as reported previously 12. 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.

Two novel alleles coding for this protein family are described in Additional File 2. The gene coding for this protein has a 3 exon structure111 on supercontig1.116. 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|61742035112 probably represents a misannotated protein with a DNA frame-shift error. Three iterations of PSI-BLAST113 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 (NCBI) (which contains all proteome versions released by ENSEMBL). 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 35 cycles of amplification (Figure 5). The function of any member of this protein family is unknown.

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 114. 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 30 ag (for gi|14423642115 and gi|18568322116) in the 2D gel experiment shown in Figure 4. The two proteins are coded by an inverted tandem repeat in supercontig1.464 separated by only 363 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 (An. gambiæ, An. stephensi, An. albimanus, An. dirus), only one polymorphic gene exists for this protein family per genome.

Two different transcripts117 in Æ. ægypti are possibly obtained from the same genomic region coding for the basic (pI = 9.4) salivary protein AE-236 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 libraries118. 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 30 kDa protein family. Four iterations of Psi-blasT119 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 7 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 (marked I in Figure 8) with three members of the family per Ædes species (two 30-kDa genes, one 29-kDa gene, plus alleles) and one member (plus alleles) 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-236 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.

Additional File 2 also includes 14 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; 13 of these are novel. Among this class of polypeptides, nine were analyzed by RT-PCR (Figure 5 and Table 2), and seven are significantly underrepresented in the non-sialotranscriptomes, as follows: AE-212120, which is similar to Drosophila and Culicoides proteins of unknown function and was ubiquitously expressed by RT-PCR experiments; two alleles (AE-165121 and AE-163122) coding for basic (pI = 9.8) 29-kDa proteins containing 4 putative galactosylated Ser/Thr and similar to Culex and Culicoides123 salivary proteins; three polypeptides whose expression appeared gland specific, as suggested by RT-PCR (AE-196, which is similar to the An. gambiae gSG8124 salivary protein; gi|61742023, which is similar to tryptophan-rich salivary proteins of Culex125; and AE-209, similar to another Culex salivary protein126). AE-225 (ubiquitously expressed by RT-PCR experiments), is weakly similar to proteins varying in size from 150–180aa residues from Anopheles127 and Drosophila128. Two additional transcripts, AE-937 and AE-752, were expressed solely in the adult female SG by RT-PCR. The function of these proteins remains to be investigated.

Two single exon129 genes separated by ~20 kb in supercontig1.15 code for proteins with signal peptides and mature mass of 62–63 kDa. Transcripts for these genes are significantly overrepresented in the sialotranscriptome and are shown to be adult female SG specific by RT-PCR (Figure 5 and Table 2). They are similar to homologous salivary protein sequences seen in Ae. albopictus130 and, to a much smaller degree, to rhoptry proteins of Plasmodium. Repeated Leu and Glu residues provide similarities to myosin131, 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 62 k by 2D gel electrophoresis MS/MS (Figure 4).

Seven transcripts coding for related proteins were found mapping to supercontig1.92. After locating the corresponding genomic regions, these 7 transcripts were annotated as truncated forms or alleles of three genes found as a tandem repeat (Figure 9). We additionally found one possible related gene in the most distal region of the 34-kDa cassette (Putative_34 kDa in Figure 9). Except for the first gene on the cassette, which codes for a 16-kDa protein and has two exons132, the remaining genes are single exonic133 and code for proteins of ~34 kDa. The two central genes, each with a single exon, are abundantly expressed as evidenced by MS/MS sequencing of tryptic digested bands (34k1 and 34k2 in Figure 4). 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 (Figure 5). Protein products of these genes match significantly only Ae. albopictus proteins134. 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 (not shown). The single-exon nature of most members of this gene family and their uniqueness among Metazoa points to a horizontal acquisition of this gene.

Two genes coding for proteins of ~30.5-kDa (not to be confused with the 30 kDa/GE-rich protein family) are found as a tandem repeat on supercontig1.280. The gene coding for gi|61742033 is abundantly expressed as evidenced by MS/MS of tryptic digested band labeled 30.5 in Figure 4. These proteins are similar only to homologues135 found in Ae. albopictus136. 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.

Two genes137 having 80% sequence similarity and coding for mature peptides of 8.5 and 9.5 kDa are found as a tandem repeat in supercontig1.18. They are similar only to salivary peptides of Ae. albopictus138. Both genes are significantly overexpressed in sialotranscriptomes. RT-PCR suggests that these genes occur in both male and female SG.

Additional File 2 lists an additional 11 full-length transcripts originating from 10 genes coding for proteins found to date only in the genus Ædes. Six of these genes have overrepresentation in the sialotranscriptome, as follows: AE-376139, AE-156140, gi|18568314141, gi|18568282142, AE-211143, and AE-214144. RT-PCR in 10 of the 11 transcripts show enrichment or female specificity for 5 transcripts and ubiquitous expression for 2 genes, while 3 appear to be transcribed in both male and female SG (Figure 5, Table 2, and Additional File 2). Their function is unknown.