The first D7 protein was cloned from a cDNA library from adult female Ae. aegypti SGs. It had an appropriately cryptic name because, at the time, it did not match other known proteins and its function was thus unknown 22. Additional members of this family were later described in An. gambiae, other mosquito species, and also in sand flies 112324. In these insects, salivary D7 proteins are encoded by multiple genes, and short and long versions of this protein family were recognized. The D7 protein family was then identified to be a member of the odorant-binding protein (OBP) superfamily 25, the long versions containing two and the short versions containing one OBP domain. Because insect OBP are known to bind and carry lipophylic compounds such as odorants and pheromones, the potential function of D7 proteins was proposed to be related to binding one or more agonists of hemostasis and thus help blood feeding 23. This prediction was confirmed when the short D7 proteins from An. gambiae and the carboxy terminal domain of the long D7 of Ae. aegypti were found to bind biogenic amines with high affinity 26. More recently, the amino terminal OBP domain of a D7 long form of Ae. aegypti was shown to bind peptidic leukotrienes with high affinity. The crystal structures of a short D7 protein from An. gambiae and a long D7 protein from Ae. aegypti revealed that the D7 OBP domains have seven alpha helices, two more than the canonical OBP family 27. In addition to these inflammatory agonist-binding functions, a short D7 protein from An. stephensi, named hamadarin, was shown to inhibit bradykinin formation by inhibiting the FXII/Kallikrein pathway 28.

An. gambiae has three genes coding for long D7 proteins and five coding for the short proteins, arranged in a single contiguous gene cassette in chromosome 3R 13. We will refer below to these proteins from An. gambiae by the transcriptional order that their genes appear in chromosome 3R. Twelve An. darlingi proteins exhibiting sequence similarity to proteins from the D7 family were identified (Table 2 and Supplemental Table S2). These include five pairs that are more than 95% identical to each other and are probably alleles. Accordingly, at least six unique products from the D7 family are identifiable in the An. darlingi salivary transcriptome. The alignment and phylogram of these protein sequences with all the D7 protein sequences of An. gambiae reveal i) the existence of An. darlingi proteins that are uniquely shorter, indicated by the bar above the alignment (Fig. 2A), which form a robust clade named 'Short AD clade' in Figure 2B. This clade is most closely related to the short D7 proteins 1 and 4 from An. gambiae (Fig. 2B), as indicated by strong bootstrap support; ii) homologues of An. gambiae short proteins 2 and 3 are identifiable (indicated as s2/s3 homologue in Fig. 2B), as well as the ortholog of the fifth short protein of An. gambiae; and iii) AD-118 represents an An. darlingi long D7 protein that is related to An. gambiae long D7 proteins 1 and 2.

AD-1 and AD-3, which possibly derive from a polymorphic gene, are similar to the D7s2 and D7s3 of An. gambiae. These proteins have in common a similar size as well as being the most transcribed D7 proteins in both species 13. AD-1 and AD-3, but not the other An. darlingi D7 sequences, share an amino acid (aa) pattern, included in a cysteine framework, that are known from crystal structure to make contact with biogenic amines 2729. The high transcription of these gene products is in line with the large amounts of protein needed to scavenge biogenic amines that accumulate to the order of one micromolar in the host tissues 26, suggesting these An. darlingi proteins, likewise their An. gambiae homologues, function as biogenic amine scavengers.

D7s1 from An. gambiae, the homologue of An. stephensi hamadarin 28 has an alkaline pI of 9.22, contrasting with the neutral or acidic pI of the remaining short D7 proteins. To the extent this basic pI is associated with hamadarin function, it is worth noting that AD-81 and AD-31 (Fig. 2) also have pIs above 8.5, but not the more distantly related AD-97. These three An. darlingi proteins are members of the novel short AD clade (Fig. 2B), which shares the same tree branch where D7s1 from An. gambiae are located, suggesting they could have a similar function as hamadarin.

This protein family, found exclusively in the SGs of adult female mosquitoes, was first identified as a salivary antigen in Ae. aegypti 30 and later found in salivary transcriptomes and proteomes of both culicine and anopheline mosquitoes 46789133132, where it was named GE-rich protein. Proteomic work also indicated that this is one of the most abundant proteins in the SGs of mosquitoes. Its gene promoter has been used to specifically drive abundant gene expression in the SGs of transgenic mosquitoes 33. More recently, proteins of this family from Aedes and Anopheles were shown to prevent platelet aggregation by collagen 3435, indicating conservation of function after the split of the Culicidae into the culicines and anophelines, ~150 MYA 17.

Analysis of the sialotranscriptome of An. darlingi allowed the identification of 8 protein sequences from this family, all represented by 2–17 ESTs found in the library. These protein sequences most probably reflect alleles from a single polymorphic gene, as they all share at least 95% identity 36. This degree of polymorphism is paralleled in the An. darlingi D7 proteins but is greater than that determined in sialotranscriptomes of other mosquitoes. Possibly this high degree of sequence variability reflects our material deriving from field-caught insects, whereas previous sialotranscriptomes were made with more genetically uniform mosquito colonies.

Alignment of all known members of this family, excluding those that are more than 95% identical and of the same species, shows their structure clearly to be dominated by three domains 34: the signal secretion peptide, a Gly/Glu-rich region, and a more conserved and organized region where the block T-x(29,30)-Q-x(5)-P-x(13,15)-I-x(2)-C-F-x(20)-C-x(8,10)-C-x(19,21)-C can be identified (Fig. 3A). This block was used by the seedtop program http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/seedtop.html to search over 6 million sequences of the NR database, only retrieving mosquito proteins. The phylogram (Fig. 3B) obtained from the alignment produces strong bootstrap support for three genus-specific clades, containing three genes for Ae. albopictus and Ae. aegypti, two for Culex quinquefasciatus, and one for each anopheline, An. stephensi, An. funestus, An. gambiae, An. darlingi, An. albimanus, and An. dirus. The An. darlingi protein groups, as expected, with An. albimanus, another American species from the Nyssorhynchus subgenus. Near the amino terminal of the mature sequences, the Nyssorhynchus-derived 30-kDa antigen/GE-rich sequences have an RGD motif as pointed out before for the An. albimanus sequence 32; this triad is not found in similar proteins of other mosquitoes. RGD-containing peptides are commonly found in snake venoms 37 and tick saliva 38, and the motif itself is usually found surrounded by two relatively close Cys groups that allow the RGD to be at the edge of a loop. This conformational feature permits the aa of the RDG motif to interact with integrins, disrupting platelet aggregation 39. It is unknown, however, whether the RGD domain present in the 30-kDa antigen/GE-rich proteins of Nyssorhynchus mosquitoes is structurally capable of interacting with integrins.

The salivary anticlotting agent of An. albimanus, named anophelin, was previously characterized as a short acidic peptide with strong thrombin inhibitory activity 4041. Despite extensive sequencing of the salivary transcriptomes of many hematophagous arthropods, similar sequences are found only in sialotranscriptomes of anopheline mosquitoes. Two similar An. darlingi cDNAs, probably corresponding to alleles of a single gene, were identified. Conceptual translation of the gene results in acidic peptides of 6.3 kDa and pI of 3.9, which are 86% identical to An. albimanus anophelin 42.

The gSG7 family is also unique to anophelines. In An. gambiae, it has two genes coding for gSG7 and gSG72, both of which are highly enriched in female SGs 13. More recently, the An. stephensi homologue was determined to inhibit kallikrein and production of bradykinin, a pain-producing substance 43. Four putative alleles representing the homologue(s) of gSG7/Anophensin in An. darlingi were identified. These An. darlingi SG transcripts, though, have no more than 45% identity to the An. gambiae gSG7 and An. stephensi anophensin 44.

The Kazal domain is ubiquitously found in proteins of metazoan organisms and, accordingly, peptides containing this domain have been identified in studies of sialotranscriptomes and proteomes of tabanids 4546, triatomine bugs 4748, Culicoides sonorensis 49, and mosquitoes 478. In Ae. aegypti and Ae. albopictus, the transcripts encoding Kazal domain proteins were ubiquitously expressed in all major organs analyzed, suggesting their function was not specific to blood feeding 47. Kazal domain peptides have also been isolated and biochemically characterized from the midgut of triatomines, where they act as anticlotting agents 505152, and from leech saliva, where they inhibit mast cell tryptase and plasmin 535455. Midgut transcriptomes of sand flies have also uncovered transcription of genes encoding peptides of this class 5657. In addition to their classical function as protease inhibitors, Kazal domain-containing peptides were identified as the salivary vasodilator of the horse flies Hybomitra bimaculata and Tabanus yao 4546. In An. darlingi, transcripts coding for three peptides with Kazal domain were found, yielding predicted mature MW of 7.2–8.1 kDa and basic pI (8.3–9.4). AD-417 and AD-257 best match An. gambiae peptides when subjected to blastp against the NR database, albeit at only 45% 58 and 44% identity 59. AD-350 best matches Aedes and Culex peptides at 47% and 51% identity 60. The function of these salivary peptides in mosquitoes remains to be discovered.

Serine- and threonine-rich proteins are commonly found in sialotranscriptomes. These proteins are generally modified post translationally, and their mature forms have N-acetyl galactosamine residues, typical of mucins 61. They probably have a function to lubricate the food canals and may also have antimicrobial function. Several protein families are represented in this group, including those previously described as SG3, gSG10, and 13.5-kDa families. Peritrophins are proteins with a chitin-binding domain that are often found in sialotranscriptomes and may be related to the maintenance of the structure of the mouthparts and/or salivary canal.

The SG3 family in An. darlingi is highly expressed, four proteins of which account for 90 ESTs found in the cDNA library. They may be alleles or splice variants of a single gene 62, containing 29% to 32% Ser + Thr and over 47 predicted galactosylation sites in a mature 17-kDa protein framework 63. The An. darlingi SG3 has similarities only to other anopheline salivary proteins, having only 46% identity and 56% similarity to the closest relative, from An. funestus 64. Compared to the Old World anophelines, the An. darlingi SG3 has a long GH repeat, which may confer zinc chelation capability and hence a putative antimicrobial activity for these proteins, because zinc chelation is characteristic of histidine rich antimicrobial agents that act by sequestration of this essential microbial growth factor 656667.

The gSG10 family, containing three peptides (Supplemental Table S2), is represented by mature products with MW of 18 kDa, 22% to 23% Ser + Thr, and 15–20 predicted galactosylation sites 68. They also may be products of a single polymorphic and/or differentially spliced gene 69. An. darlingi gSG10 members match both anopheline and culicine sequences of salivary origin 70, having a unique signature block 71that characterizes these distinctive mosquito proteins.

The 13.5-kDa protein family is also represented in An. darlingi by the products of two or three genes 72. Most mosquito 13.5-kDa family members have over 30 predicted galactosylation sites 73. An. funestus, An. gambiae, and An. stephensi have recognizable relatives; however those proteins show only 41% to 44% identity over most of the length of the protein to the An. darlingi 13.5-kDa products. Culicine proteins that display only conservation of the stretch of threonine residues have been identified, but they may not be true homologues.

Two other putative mucins were found, AD-11 being a hypothetical secreted peptide of predicted mature MW of 3.8 kDa, 25% Ser + Thr, and ten potential glycosylation sites. No significant matches are found with other known proteins. AD-91, on the other hand, with 20% Ser + Thr content and 20 potential O-glycosylation sites, is 71% identical to an An. gambiae protein 74 that is related to a previously identified Aedes salivary protein and to a Drosophila protein annotated in the Gene Ontology database as associated with defense response to virus 75.

A single transcript in the An. darlingi sialotranscriptome codes for a peritrophin with a typical chitin-binding domain 76 and 69% sequence identity to an An. gambiae protein annotated as peritrophin A 77, which was cloned from the mosquito midgut 78.

The SG3, SG10, and 13.5-kDa families were found abundantly expressed in sialotranscriptomes of adult male An. gambiae 78, indicating their function is likely not related specifically to blood feeding.

Enzymes associated with both blood (apyrase and peroxidase) and sugar (amylase and maltase) feeding are known to occur in mosquito saliva; accordingly, their corresponding transcripts have been found in mosquito sialotranscriptomes. Serine protease-encoding transcripts also are regularly found, but their proposed functions in helping blood feeding by interacting with host proteins or as participants in immune proteolytic cascades have not been validated.

Apyrase, which hydrolyses ATP and ADP to AMP and orthophosphates, has been a ubiquitous finding in the saliva of blood-sucking arthropods, where it destroys these important agonists of inflammation and platelet aggregation 279. Mosquitoes have co-opted the 5' nucleotidase family to achieve this function 808182. Two genes of this family are expressed in the SGs of An. gambiae 13, named putative 5' nucleotidase and salivary apyrase, although both may function redundantly as apyrases. The sialotranscriptome of An. darlingi presents evidence for the two orthologues, IS07-44, a full-length orthologue of the salivary 5' nucleotidase of An. gambiae 83, to which it is 66% identical, and AD-101, which is a 5' truncated clone best matching the An. gambiae salivary apyrase 84.

A peroxidase was previously identified as the vasodilator for norepinephrine-induced aortic contractions found in An. albimanus SGs 8586. AD-573 encodes the full-length sequence of an An. darlingi salivary peroxidase that is 86% identical to An. albimanus and 52% identical to An. gambiae salivary peroxidases 87. This type of salivary vasodilator is so far unique to anopheline mosquitoes.

Maltase and amylases, as well as their transcripts, have been regularly found in the saliva and sialotranscriptomes of mosquitoes 88899091. The first cloned gene from the SGs of any mosquito was actually a member of this family 92. Ae. aegypti and An. gambiae express both genes in their SGs. Transcripts coding for both enzymes were found in the sialotranscriptome of An. darlingi [see additional file 1]. The full-length sequence for the orthologue of An. gambiae salivary maltase (68% identity) 93 is presented in Supplemental Table S2.

Transcripts coding for at least two different serine proteases were found in An. darlingi sialotranscriptome [see additional file 1]. Supplemental Table S2 presents a truncated sequence of a CLIP domain serine protease expressed in An. darlingi SGs, 86% identical to the An. gambiae closest match 94.

Antimicrobial peptides, lysozyme, and pathogen pattern recognition polypeptides are commonly found in the sialotranscriptome of blood-sucking arthropods. Among the AMPs found in the sialotranscriptome of An. darlingi, a gambicin 95, a defensin 96, and three different cecropins 97 are described in their full-length condition. A peptidoglycan recognition protein, 94% identical to an An. gambiae protein 98, is also reported as a full-length protein. Additionally, this study [see additional file 1] provides evidence for An. darlingi transcripts coding for C-type lectins and ficolins, and an odd transcript having a full PMEI Pfam domain 99 normally found in plant proteins associated with inhibition of microbial pathogens' pectin methyl esterase. Two similar lysozyme cDNAs, probably products of alleles, are also described as full-length polypeptides, matching 57% identity to the closest An. gambiae protein 100. Another identified lysozyme, contig 443 101, corresponds to a previously described salivary An. darlingi lysozyme 15. The occurrence of multiple lysozymes in the An. darlingi sialome is not surprising, as two lysozymes are expressed in the An. gambiae SGs 13.

With less certainty, we include in the immunity-related products the full-length sequence for a Gly-His-rich peptide that might have antimicrobial function by zinc chelation, as explained above. This protein matches a C. quinquefasciatus salivary peptide that also contains Gly repeats and a poly His in the amino terminus 102.

This is a ubiquitous protein family found in animals and plants 103 and in all sialotranscriptomes of blood-sucking Diptera analyzed so far. The function of these proteins in mosquito saliva is not known, although they were implicated in a proteolytic function in the venom of the marine snail Conus textile 104, in toxic functions in the saliva of a venomous lizard and snake venoms 105106107108109, and in an antifungal function in plants 110. Remarkably, a member of this family acquired a typical RGD domain surrounded by Cys residues and acts as a main platelet aggregation inhibitor in the horsefly Tabanus yao 46. Several genes from the AG5 family are transcribed in the SGs of mosquitoes, including some specific to the adult females and thus possibly associated with a specific function in blood feeding 4713. We present evidence, in the form of full-length transcripts, for the expression of at least two members of the AG5 family in An. darlingi SGs 111. AD-38 matches with 67% identity the putative gVAG protein precursor of An. gambiae 112, a transcript enriched in the adult female SGs when compared with expression in other tissues 13. AD-430 matches An. gambiae AG5-related 2 protein 113, which was shown to be ubiquitously expressed in adult female tissues 13. The function(s) of this protein family in mosquitoes remain to be determined.

Transcripts coding for the gSG5 protein 114 were first discovered in the SGs of An. gambiae and shown to be exclusively expressed in the adult female SGs 13115. This protein produces weak similarity to a salivary protein of Ae. aegypti 116 and better similarity to other Aedes 117 and Culex proteins 118, indicating this is a mosquito-specific protein. Six transcripts coding for this protein were found in the sialotranscriptome of An. darlingi. AD-196 is 46% identical to the An. gambiae orthologue and only 26% and 23% identical to the culicine proteins 119. The function of this mosquito-specific protein remains unknown, but its tissue- and sex-specific expression profile suggests it is possibly related to blood feeding.

The gSG8 is a highly divergent family, with members only from An. gambiae and Ae. aegypti 120. Alignment of the three sequences displays a conserved motif L-C-W-A-x-K-x(2)-P-T-A-x(6)-C-x(5)-K, which might help identify new members of this family. In An. gambiae, this protein is specifically expressed in female SGs 115, suggesting a likely role in blood feeding.

AD-216 and AD-217 represents two similar proteins deducted from two and three ESTs, respectively. They may represent splicing variants or alleles of the same gene 121. The predicted mature peptides have 11.2 kDa and solely match proteins found in other mosquito sialotranscriptomes or other hypothetical mosquito proteins 122. The basic tail name derives from a conserved Lys-X-X-Lys or Lys-X-X-Arg found in the carboxyterminus of proteins derived from the genus Aedes but lacking in the anopheline sequences. The alignment indicates a conserved backbone and the absence of cysteine residues, from where the block pattern L-x-H-x-L-x-Y-L-x-D-x(17,18)-A-x(2)-Y-x(3)-A-x(3)-G can be deduced (Fig. 4A). The derived phylogram (Fig. 4B) follows the expected mosquito phylogeny. Ae. aegypti transcripts coding for the basic tail peptide were enriched in adult female SGs 7.

AD-476 represents the peptide sequence of a mature protein of 4.1 kDa having significant similarities only to other polypeptides found previously in culicine mosquito sialotranscriptomes or predicted proteomes of mosquitoes 123. This is the first time a protein of this family is found in an anopheline sialotranscriptome. Alignment and phylogram of the mature predicted peptides shows that Ae. aegypti and C. quinquefasciatus have two such peptides, those of Anopheles matching the slightly smaller version (Fig. 5A). The derived phylogram indicates two clades grouping the short and the large forms. In Ae. aegypti, transcripts coding for a member of this family were shown to be enriched in the adult female SGs 7.

The sialotranscriptome of Ae. aegypti identified a protein named proline-rich salivary secreted peptide 124, close homologues of which were never found in other sialotranscriptomes. Transcripts for this protein were found exclusively on the adult female SGs of Ae. aegypti, indicating a function related to acquisition of the blood meal 7. The sialotranscriptome of An. darlingi provided three ESTs, which when assembled derive the sequence AD-267, matching this Aedes protein at 47% identity 125 and also, weakly, a smaller region of a salivary protein from An. stephensi of the same size. AD-267 was subjected to psiblast analysis against the NR database retrievieng only sequences from Ae. aegypti, which converged after two iterations. The presence of AD-267 in An. darlingi, its homology to the Ae. aegypti protein, and its absence in An. gambiae suggest that the gene for this family existed in the ancestor of culicines and anophelines but was lost or modified beyond recognition in Culex and the Cellia subgenus of Anopheles.

The first 41.9-kDa family member was characterized in sialotranscriptome of Ae. aegypti and later found in C. quinquefasciatus and in Ae. albopictus 47810. It has never been found in any anopheline sialotranscriptome, nor does it have any similar protein predicted from the An. gambiae genome 126. AD-114, however, produces similarities to 41.9-kDa family members when subjected to blastp analysis against the NR database 127. The blast results interestingly retrieves other salivary proteins from hematophagous Diptera from the NR database, such as gSG10, gSG9, and other mucins, despite having itself only three potential galactosylation sites. The alignment of the An. darlingi protein with the 41.9-kDa proteins from Ae. aegypti and C. quinquefasciatus shows extensive similarities over the whole length of the sequences, including a conserved cysteine framework, despite having less than 30% identity with the culicine proteins (Fig. 6). AD-114 thus appear to be a "missing link" joining previously thought unrelated salivary protein families from Culicines and Anophelines. To further investigate this possibility, we used psiblast to search AD-114 against the NR database, retrieving mostly proteins found before in sialotranscriptomes of blood-sucking Diptera 128, including Culicoides 49 and sand flies 129130. In addition to the known 41.9-kDa members from culicines, the anopheline proteins annotated as gSG10 and gSG9 are also retrieved, as are a group of proteins annotated as salivary mucins from mosquitoes, including the non-bloodfeeding species Toxorhynchites amboinensis 131. Exceptionally, two bacterial proteins are retrieved, as well as one from the wasp Nasonia vitripennis. The alignment of the proteins from Diptera plus the two bacterial proteins by the Clustal tool does not reveal any region of common conservation among all proteins (not shown), but the derived bootstrapped phylogram (Fig. 7) is informative. Strong support is obtained for four clades, as indicated in Figure 7. The first clade includes sequences from both anopheline and culicine mosquitoes annotated as gSG10, gSG9, and mucins, together with the An. darlingi sequence. A second clade includes Culex and Aedes proteins annotated as mucins. This second clade roots with strong bootstrap support to the previous clade. A third clade includes Aedes proteins annotated as 41-kDa protein, or a short version, annotated as 30.3-kDa protein. This clade also roots strongly with the two previous clades. The sole C. quinquefasciatus sequence shown in Figure 7 (gi|170045863), the 41.9-kDa basic salivary protein, does not group significantly with any other sequence. Finally, a fourth clade groups together the bacterial and sand fly proteins. This clade does not root with strong bootstrap support to the previous clades. The presence of the bacterial proteins in this clade is puzzling, and suggests that the Nematocera proteins could have derived from bacterial contaminants. However, the proteins deriving from Ae. aegypti, C. quinquefasciatus and An. gambiae map to assembled chromosomes or supercontigs, and their respective genes contain introns indicating they are of eukaryotic origin. Together, these results support the argument that the 41.9-kDa protein family of mosquitoes has a common salivary ancestor before the split of anophelines and culicines, being recognized in An. darlingi by AD-114; in the Cellia subgenus, the 41.9-kDa protein family has evolved to produce shorter proteins, the subfamily members of the gSG10 and gSG9 families. Sand flies express related salivary proteins that might have been acquired by convergent evolution or share a distant common ancestor that can no longer be recognized with the available sequences.

Six genes coding for proteins of this unique protein family were found in An. gambiae salivary transcriptomes 1112115, four of which are located as a contiguous gene cluster 132 in chromosome X 13. Remarkably, all these genes are uniexonic, unusual for eukaryotic genes coding for these relatively large proteins, attaining a mature molecular weight above 40 kDa, suggesting its acquisition as horizontal transfer. This gene family appears to be specifically associated with SG function. The transcripts coding for the Trio, SG1, and SG1b proteins appears to be exclusively expressed in the female SGs, while SG1-like3 and gSG1-2 and gSG1a are enriched in the female glands but also present in lower amounts in male glands and not observed in other tissues 13. When these proteins were subjected to blastp against the NR database, only other anopheline sequences are retrieved. Sixty-three ESTs were found in the An. darlingi sialotranscriptome coding for proteins of this family, from which six full-length clones were sequenced. Of these six sequences, two possibly derive from alleles or splice variants 133. When full-length protein sequences from all known members of this family are aligned by the Clustal tool, very few conserved aa are identified (Fig. 8A); however, the deduced phylogram show strong bootstrap support for five clades (Fig. 8B), named for the An. gambiae proteins, as follows: Clade SG1/SG1a contains these two proteins from An. gambiae and also one sequence each from An. stephensi, An. dirus, and An. darlingi. Clade SG1-like3 contains two sequences from An. darlingi that could be the result of a recent gene duplication or polymorphism and splice variation 134. These two sequences cluster with strong bootstrap support, as expected, with the sole sequence from An. albimanus. The Trio clade also has AD-153 from An. darlingi. The clade SG1-2 is the only clade not having An. darlingi representatives. The function of these proteins remains to be determined.

The SG2 protein was deduced from salivary An. gambiae cDNAs and shown to be expressed in female glands and adult males but not in other tissues 11. It derives from a single gene in chromosome 2L and is abundantly transcribed in sialotranscriptomes of male An. gambiae 135. Related, but very divergent, sequences were obtained solely from salivary transcriptomes of other anopheline species 6. The sialotranscriptome of An. darlingi indicates that at least two different genes exist coding for proteins of this family. One gene codes for mature proteins of 8.5 kDa, from which four alleles or splice variants are derived 136. A second gene may have produced another five different alleles or splice variants coding for shorter (5.6- to 6.1-kDa) peptides 137, but it is more likely that these derive from two closely related genes. Comparison of these proteins with other anopheline sequences displays sequence identities varying from only 26% 138to 31% 139. Because this protein family is expressed in both male and female An. gambiae 11135, and due to its relatively small size, it may display antimicrobial function.

The hyp 15 and hyp 17 proteins, previously identified in sialotranscriptomes of An. gambiae 12, have alkaline pI and ~4.7 kDa. Their genes reside as tandem repeat in chromosome X and are preferentially expressed in adult female SGs 13. Homologues were additionally found in An. stephensi and An. funestus. The An. darlingi sialotranscriptome presents evidence of three transcripts that may derive from splice variants from a single gene 140, which are 41% and 39% identical to the An. funestus and An. gambiae homologue 141.

In An. gambiae, the genes coding for the hyp 8.2 and hyp 6.2 proteins are found as a tandem repeat in chromosome arm 2L. These proteins have mature molecular weight of 6–9 kDa, do not have sequence similarity, and are grouped together solely by virtue of being chromosomal neighbours. Transcripts coding for these two polypeptides are similarly enriched in An. gambiae adult female SGs 13. An. stephensi and An. funestus also have members of these protein families. In An. darlingi, two quite divergent protein sequences 142 deduced from the sialotranscriptome are similar to hyp 8.2 143, and one is similar to hyp 6.2 144.

An. darlingi protein AD-269 has a predicted molecular weight of 6.5 kDa and matches 145 the carboxyterminus of a salivary peptide named hyp 5.6 previously described in An. gambiae sialotranscriptome 13. Members of this family have not been found previously in other sialomes. In An. gambiae the transcript coding for hyp 5.6 was ubiquitously transcribed, suggesting a housekeeping or antimicrobial role.

A protein cryptically named hypothetical protein was previously identified in a cDNA library of An. gambiae 115, but homologues were never found in other sialotranscriptomes of either anopheline or culicine mosquitoes. This An. gambiae protein produces matches to other unrelated sequences in the NR database by virtue of repeated acidic amino acids. The sialotranscriptome of An. darlingi produced 60 transcripts matching this An. gambiae protein, distributed into six putative protein sequences deriving from possibly two genes 146, of which AD-18 represents a shorter form of the family (Fig. 9). The five remaining deduced sequences may result from alleles 147. These proteins have predicted mature molecular weight of 14–17 kDa and pI of 4.2. They are 41% 148 to 50% identical 149 to the An. gambiae homologue. Alignment of two of the An. darlingi sequences with the An. gambiae homologue identifies a region of Ser [Asp/Glu] [Asp-Glu] repeats (identified with a bar labelled I in Fig. 9) and a region of two repeats WIRRP in the An. gambiae sequence (identified with a bar labelled II in Fig. 9), which provides a name for the family.

Two An. darlingi protein sequences, never before evidenced in mosquito sialotranscriptomes, are described here with clear signal peptide indicative of a secretion. These are AD-136, which significantly matches only hypothetical proteins of An. gambiae, Ae. aegypti, and C. quinquefasciatus 150, and AD-119, which has no significant matches to any known protein in the NR database. Seven and 15 transcripts were found coding for each protein, respectively. AD-136 has an allele 151, AD-138, derived from two transcripts.

In a previous sialotranscriptome analysis of An. gambiae, 92 transcripts from a total of 4,066 13 coded for a protein named gSG6 115, orthologues of which were found in An. stephensi and An. funestus sialotranscriptomes 152. Considering that we have sequenced in the present work 2,371 ESTs from An. darlingi, some 53 ESTs would have been expected for this protein. None were found, suggesting this family to be specific for the Cellia subgenus. Similarly, the related An. gambiae proteins named hyp 10 and hyp 12 153 had 37 and 12 corresponding ESTs, but none were found in the An. darlingi cDNA library, also suggesting this family to be Cellia-specific.