An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae

The salivary glands of blood-sucking arthropods express a varied mixture of anti-hemostatic and immunomodulatory components that help the arthropod to take, or to find, a blood meal (Ribeiro,1995). In the case of mosquitoes, only the adult female is hematophagic, whereas both male and females take sugar meals. Perhaps for this reason, adult mosquitoes also have salivary glycosidases(Grossman et al., 1997; Marinotti et al., 1990) and anti-microbials (Rossignol and Lueders,1986) that may prevent bacterial growth in the sugar meal stored in the mosquito crop.

The `classical' process of learning the function of salivary gland products in vector arthropods starts with the discovery of a biological activity in crude homogenates, then isolation of the protein and, finally, description of the DNA sequence coding for the protein primary structure. Recent advances in transcriptome techniques led to the reversal of these steps in such a way that the primary sequence of many putatively secreted salivary proteins are now known; but for only a minority of these do we yet know the function and even whether they are really secreted (Ribeiro and Francischetti, 2003). In the case of the mosquito Anopheles gambiae Giles, the main vector of malaria in Africa,previous transcriptome analysis of nearly 500 expressed sequence tag (EST) and signal sequence trap methods was used to identify genes expressed in the adult female salivary glands (Arca et al.,1999; Francischetti et al.,2002; Lanfrancotti et al.,2002). Accordingly, a combined non-redundant (NR) set of 40 proteins has been proposed to be of a salivary secretory nature in An. gambiae; we can assign a function based on experimental evidence for fewer than 10 of these.

The recent elucidation of the genome of An. gambiae associated with high-throughput transcriptome analysis facilitates further gene discovery. In the current paper, we present the analysis of an additional set of 2396 salivary gland cDNA sequences (total of 3087 compared with previous set of 691 clones), resulting in the discovery of 33 new salivary gland proteins. An NR catalogue including 72 transcripts – of which 71 code for proteins of a putative secretory nature – is presented and discussed. It should be helpful in designing experiments to determine the function for the majority of these transcripts. To this end, we analyzed the tissue and sex specificity of 88 transcripts and found that 27 are either exclusively expressed or enriched in the salivary glands.

An. gambiae (G3 strain) salivary gland mRNA was isolated from 80 salivary gland pairs from adult females at days 1 and 2 after emergence using the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA). A cDNA library was constructed, and randomly selected cDNA clones sequenced as previously described (Francischetti et al., 2002).

ESTs were trimmed of primer and vector sequences, clusterized and compared with other databases as described before(Valenzuela et al., 2003). The BLAST tool (Altschul and Gish,1996) and CAP3 assembler were used(Huang and Madan, 1999), as well as the ClustalW (Thompson et al.,1994) and Treeview software(Page, 1996). O-glycosylation sites on the proteins were predicted with the program NetOGlyc(http://www.cbs.dtu.dk/services/NetOGlyc/)(Hansen et al., 1998). We submitted all translated sequences (starting with a Met) to the Signal P server (Nielsen et al., 1997)to detect signal peptides indicative of secretion. For visualization of EST on the An. gambiae genome, the EST and cluster sequences were mapped to the An. gambiae genome using the Artemis tool(Berriman and Rutherford, 2003)after downloading the GenBank-formatted files for the An. gambiaechromosomes from Ensembl(ftp://ftp.ensembl.org/pub/current_mosquito/data/flatfiles/genbank/). Because the files for each chromosome or chromosome arm are partitioned into several different files, a program written in Visual Basic was used to read the GenBank-format files to obtain a single fasta-formatted file for each chromosome or chromosome arm and a uniform rather than relative location for each gene, producing a feature file that could be read by Artemis. Accordingly, Artemis could read a single flat file and a single set of features for each chromosome or chromosome arm instead of breaking up each chromosome into several dozen pieces. The unique fasta files for each chromosome were, in turn, broken into 30-kb fragments with 5 kb from previous sequence to speed BLAST analysis. The EST and contigs were compared with this fragmented-sequence genomic database by blastn(Altschul et al., 1997) and the output transformed to a file compatible with Artemis using a program written in Visual Basic. Sequence annotation was done with the help of AnoXcel(Ribeiro et al., 2004).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 20 pairs of homogenized An. gambiae adult female salivary glands was performed using 1 mm-thick NU-PAGE 4% to 12% gels (Invitrogen). Gels were run with MES buffer according to the manufacturer's instructions and the proteins transferred to a PVDF membrane. The membrane was then stained with Coomassie Blue in the absence of acetic acid; visualized bands (including a negative-stained band) were cut and subjected to Edman degradation using a Procise sequencer (Perkin-Elmer Corp., Foster City, CA, USA). More details can be obtained in a previous publication(Francischetti et al., 2002). To find the cDNA sequences corresponding to the amino acid sequence –obtained by Edman degradation of the proteins transferred to PVDF membranes from PAGE gels – we wrote a search program (in Visual Basic) that checked these amino acid sequences against all possible reading frames of each cDNA sequence obtained in the mass sequencing project. For details, see Valenzuela et al. (2002b).

Salivary glands were dissected from 1- to 4-day-old adult females, frozen in liquid nitrogen and stored at –80°C. Total RNA was extracted from dissected glands, carcasses (i.e. adult females from which salivary glands had been dissected) and adult males using the TRIZOL reagent (Invitrogen) and treated with RNase-free DNase I. DNase-treated total RNA (50 ng) was amplified using the SuperScript one-step RT–PCR system (Invitrogen) according to the manufacturer's instructions. Typically, reverse transcription (50°C,30 min) and heat inactivation of the reverse transcriptase (94°C, 2 min)were followed by 35 PCR cycles: 30 s at 94°C, 30 s at 65°C, 1 min at 72°C. For a subset of primer pairs, the annealing temperature was lowered to 55–60°C for optimal amplification. Twenty-five cycles were used for the amplification of the product based on the ribosomal protein S7 mRNA(rpS7) to keep the reaction below saturation levels and to allow a more reliable normalization. The sequences of the oligonucleotide primers used for rpS7 amplification were: Ag_rpS7-F-5′ GGC GAT CAT CAT CTA CGT GC 3′ and Ag_rpS7-R-5′ GTA GCT GCT GCA AAC TTC GG 3′. The sequences of the other oligonucleotide primers are provided in the supplemental material. Amplification reactions were analyzed on 1.2% agarose gels stained with ethidium bromide.

Total RNA of five adult mosquitoes was extracted to prepare each sample. A total of six samples – three from non-blood-fed females and three from sugar-fed male mosquitoes – were analyzed. Isolated total RNA was processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual; Affymetrix, Inc., Santa Clara, CA, USA). Data analysis was done with the Gene Chip Operational Software (GCOS) package. Other procedures are exactly as described before(Marinotti et al., 2005). The microarray data are available at http://www.angagepuci.bio.uci.edu/.

Clones (total 3087) were included in the EST salivary database, including 691 that had been previously described(Francischetti et al., 2002). Of these, 176 EST were identified as being of mitochondrial origin, according to their match to the An. gambiae mitochondrial genome, and were not further analyzed. The combined nuclear sequences were assembled into 861 contigs and singletons (in this paper, uniformly named contigs) after clusterization of the database (see Materials and methods). To attempt a functional classification of these unique sequences, we compared them with proteome databases by blastx and with protein motifs by rpsblast (see Materials and methods). Following manual annotation of these contigs, which included the assignment of known or putative functions to the translation products, they were further divided into five categories(Table 1): housekeeping (H class) with 357 contigs and 554 sequences; secreted (S class) with 155 contigs and 1940 sequences; transposable element (T class) with three contigs and three sequences; putative bacterial horizontal transfer contigs (W class) with nine contigs and 32 sequences; and contigs coding for proteins of unknown function (U class) with 337 contigs and 382 sequences. Although the S class corresponds to only 18% of the contigs, it consists of 67% of all EST,reflecting the relatively low complexity and abundance of the secretory material of the organ, as indicated previously(Francischetti et al.,2002).

Because a significant number of contigs did not match any protein of the An. gambiae proteome set, we considered the possibility that these U class transcripts could be representing mostly untranslated (UTR) mRNA regions. The PCR-based cDNA library used in this work supposedly provides for full-length clones by use of a strategy using polydT primers and a modified polymerase (Zhu et al., 2001)when synthesizing the cDNA from RNA. The cDNA were directionally cloned into the viral vector and sequenced only in the 5′→3′ direction,because extensions from the 3′ end most often fail to cross the polyA region. Accordingly, clones coding for 5′ UTR could derive from full-length clones with unusually long 5′ UTR, because our average read is larger than 400 nucleotides (nt). Alternatively, the sequenced cDNA could correspond to the 3′ UTR of the transcripts if the polymerase fell off its template in the cDNA synthesis step or in the case of the transcript having an Sfi restriction site, which is used during library construction. When each contig position was located in the An. gambiae genome and the closest gene in the same orientation identified, we observed that 230 contigs were near the 3′ end of predicted exons, while only 51 were near the 5′ region of predicted exons. A χ² test indicates this difference to be highly significant (P<0.001). These 230 contigs containing 263 sequences, indicating that approximately 9% of the database sequences were truncated. Additionally, 567 contigs overlapped with predicted exon locations, including some that did not give significant blastx matches to the An. gambiae proteome because they contained only a few base pairs within the predicted exon. A few (13) contigs (see supplemental spreadsheet at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls,worksheet `Nuclear', column AR) matched predicted intronic regions, usually on large genes, possibly representing alternative splicing and/or the cloning of unprocessed pre-mRNA. We also observed that many contigs coded for different locations of the same gene. A non-redundant list of gene matches is provided in the supplemental spreadsheets within the worksheet named `ENSANGP list'.

Following visualization of these U-class contigs into the genome using the Artemis tool, we further excluded some potential hits to UTR either because the nearest match was too far from the gene (`too far' was considered a distance longer than the length between the start of the first exon and the end of the last exon) or because the contig was probably coding for a novel gene (25 occurrences; worksheet named `Nuclear', column AR – search for`novel' on supplemental spreadsheet). We thus arrived at 177 contigs probably located in the 3′ UTR region of predicted genes and 23 located at the 5′ UTR. We have also observed that contigs matching 3′ UTR tended to be on large genes. Indeed, the set of predicted An. gambiae genes identified by direct contig matches to an exon had an average gene length(measured from the beginning of the first exon to the end of the last exon) of 2733 nt and had 3.32 exons, while that of genes identified by their 3′UTR was more than twice as large (6112 nt) with an average of 5.07 exons. Both differences are highly significant (P<0.001) when compared by the Mann–Whitney rank sum test. It should also be considered that some of these putative UTR transcripts may code for not-yet-identified exons. Re-annotation of the database taking into consideration the 3′ UTR matches increased significantly the number of probable H-class genes while decreasing those of the U class (Table 1).

Two transcripts on our database (contigs 284 and 285; supplemental spreadsheet at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls#BM147)possibly derive from transposable elements. Their translation products are similar to those of Caenorhabditis elegans proteins annotated as CCHC-type and RNA-directed DNA polymerase and integrase and to TY elements in the GO database, and also possess rve Pfam domains indicative of reverse transcriptases. These transcripts may indicate active ongoing transposition activity in An. gambiae.

A relatively large number of transcripts (34 sequences, organized into 11 contigs, representing 1.2% of the salivary EST originating from nuclear genes)match three genes located contiguously in chromosome arm 3R that code for the putative proteins ENSANGP00000027299, ENSANGP00000027791 and ENSANGP00000029569) (Fig. 1). Investigation of nearby genes identified another instance of a possible family member (Fig. 2;ENSANGP00000026834) without EST representation in our database. When the program PSI-BLAST was used with protein sequence 29569 above for two iterations, it gave a 0.0-e value with hypothetical protein WD0513 of Wolbachia endosymbiont of Drosophila melanogaster(gi:42520378) in addition to identifying all other proteins of the cluster. Further iterations of the program retrieve many bacterial proteins annotated as belonging to the Rhs family (Hill et al., 1994). Although the automatic ENSEMBL translation predictions indicate spliced products for the transcripts coding proteins 27791 and 26834,the cDNA we sequenced did not confirm the predictions. The transcripts are not spliced and show one single large open-reading frame(Fig. 1). The likely single-exon structure of these contiguous genes and their similarity to bacterial proteins suggests that this protein family cluster arose by horizontal transfer from a bacterial genome. Because horizontal gene transfer could be mediated by transposable elements(Syvanen, 1994), we investigated whether such sequences were present in the vicinity of these genes. Indeed, two retro transposable element-like fragments, named TE5p and TE3p, flank the region containing the bacterial genes, together with five additional genes, as shown in Fig. 2. TE5p, in particular, is located very close to the 5′-most gene coding for ENSANGP00000026834 and could have originated the lateral transfer. The BLAST alignments of TE5p and TE3p with described transposons are shown in Fig. 3A,B. The Wolbachia genus consists of Rickettsia-like organisms infecting arthropods and conferring the phenomenon of cytoplasmic incompatibility (Drancourt and Raoult,1994; Sinkins,2004). Of interest, Anopheles mosquitoes are resistant to Wolbachia (Kittayapong et al.,2000; Ricci et al.,2002), and it is hypothesized here that these Wolbachia-like transcripts may underlie such resistance.

Putative H-class genes were further classified according to their possible function (Table 2). Results are available online and can be searched on the columns labeled `Class' and`Comments' (supplemental spreadsheet at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls). Not surprisingly, the most abundant gene class expressed constitutes members of the protein synthesis machinery, which, together with transcription machinery, protein modification and protein export, comprise 34% and 43% of H-class contigs and sequences, respectively. 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, Ca²⁺-ATPases and several families of solute carriers. V-type ATPases have been implicated in the secretion of saliva in Diptera (Zimmermann et al., 2003). Several transcripts coding putative receptors were also found, including G-coupled proteins (ENSANGP00000023076), a kinase associated with β-adrenergic receptors (ENSANGP00000008658) and several subunits of the NMDA/glutamate receptor family (ENSANGP00000018675,ENSANGP00000025350 and ENSANGP00000021195). These may function in the secretion signaling of the salivary glands.

Among the transcripts coding for extracellular matrix components, we highlight those coding for laminin (ENSANGP00000010745; a gene that needs to be corrected in its intron–exon borders), the heparin sulfate proteoglycan perlecan (ENSANGP00000022422) and the enzyme chondroitin N-acetylgalactosaminyltransferase, which is involved in the synthesis of extracellular mucopolysaccharides (ENSANGP00000020105). These extracellular constituents may be important for Plasmodium recognition of the salivary glands, because sporozoites are known to recognize sulfated polysaccharides (Pinzon-Ortiz et al.,2001).

Several transcripts matched genes coding for transcription factors. Table 3 lists some of interest for the specialized function of the female salivary gland, including transcription factors associated with expression of ER chaperones (XBP-1),general transcription factors, and those associated with tissue differentiation. In particular, two genes coding for Forkhead transcription factors are indicated, as well as three involved in the Hairy pathway. The Forkhead and Hairy transcription factors have been implicated in Drosophila salivary gland differentiation and salivary protein expression (Lee and Frasch,2004; Mach et al.,1996; Myat and Andrew, 2000, 2002; Myat et al., 2000; Poortinga et al., 1998). Expression of the gene coding for doublesex, which is associated with sex-specific gene expression in Drosophila(Baker et al., 1989; Baker and Wolfner, 1988), is a good candidate to explain the sexual dimorphism observed in adult mosquito salivary glands.

After identifying putative secreted proteins (supplemental spreadsheet at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls),we used this data set and the Artemis tool to identify novel proteins coded in the An. gambiae genome. Indications of secreted polypeptides were obtained with searches for the presence of signal peptides with the SignalP program (Nielsen et al., 1997)and of O-linked galactosylation sites (indicative of mucins) with the NetOGlyc program (Hansen et al.,1998). A NR set of 72 putative proteins expressed in the salivary glands of An. gambiae is presented in Table 4; it includes 71 polypeptides predicted as secretory, 40 of which have been described previously. Of these 40, seven are described now in full length. Thirty-three proteins are indicated for the first time to be expressed in the salivary glands of adult female mosquitoes. Of these 33 proteins, 29 were predicted by the ENSEMBL annotation pipeline and four are novel. Of the 29 predicted by ENSEMBL, 17 were re-annotated to fix the starting Met or stop codons.

The first member of the D7 protein family was described in the mosquito Aedes aegypti (James et al.,1991) and later found in virtually all mosquito sialotranscriptomes. Short (∼15 kDa) and long (∼30 kDa) forms are recognized. Long D7 forms also exist in sand flies(Valenzuela et al., 2002a). The function of these proteins has not been verified, although one short D7 protein from An. stephensi, named hamadarin, was shown to prevent kallikrein activation by Factor XIIa(Isawa et al., 2002). Previously, one long D7 and five short D7 proteins were known in An. gambiae (Arca et al.,2002; Francischetti et al.,2002). Table 4shows these six proteins and two additional D7 proteins, all coded from contiguous genes in chromosome arm 3R. The three long D7 genes follow each other in the forward direction, the first two having four exons, but the last having only three exons (Fig. 4). The five short-form genes follow the long D7 cassette in reverse orientation, the first four having three exons, while the fifth has only two exons. Notably, an apparently non-coding transcript (contig_709) maps just 250 nt downstream of the short D7 cassette in the reverse orientation of the gene. We speculate that this transcript may be associated with regulation of D7 expression. Combined, these genes represented 574 sequences in our database, or nearly 20% of over 3000 EST. It is also interesting to note that the last gene in each of the cassettes, i.e. D7L3 and D7r5, was the least represented in terms of number of EST, indicating that they are expressed at lower levels than their similar neighboring genes. Moreover, in comparison to the other members of the cluster, D7L3 and D7r5 differ in the number of exons, and their pattern of expression is not restricted to female salivary glands(Table 4). Evidence for the synthesis of all but one of these proteins (named D7L3 in Table 4) in the salivary glands of female mosquitoes was found by Edman degradation of bands resolved by SDS-PAGE.

Four genes coding for members of the AG5 family were identified by matching salivary transcripts. 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 ofplants)(Megraw et al., 1998). The CAP family is related to venom allergens in social wasps and ants(Hoffman, 1993; King and Spangfort, 2000) and to antifungal proteins in plants (Stintzi et al., 1993; Szyperski et al., 1998). Members of this protein family are found in the salivary glands of many blood-sucking insects(Francischetti et al., 2002; Li et al., 2001; Valenzuela et al., 2002b). These animal proteins have no known function, except for a few cases: one Conus protein was recently shown to have proteolytic activity(Milne et al., 2003), snake venom proteins of this same family have been shown to contain smooth muscle-relaxing activity (Yamazaki et al.,2002; Yamazaki and Morita,2004), and the salivary neurotoxin of the venomous lizard Heloderma horridum is also a member of this protein family(Nobile et al., 1996). Three of the four genes identified by the transcriptome are located in a cluster of genes in chromosome arm 2L, with these three genes receiving 106, 1 and 1 matches from EST (Table 4). The fourth gene, located on chromosome arm 2R, received only one EST match from our database. Of these four putative protein sequences, one is novel and another reports the full length of a previously identified salivary-expressed sequence. Further work on this gene family will be reported elsewhere (B. Arcà et al., manuscript in preparation).

This family of salivary proteins, having mature molecular masses near 44 kDa, was previously described as SG1 or gSG1(Arca et al., 1999; Lanfrancotti et al., 2002). They do not yield significant similarities by blastp to other proteins in the NCBI database except for other anopheline proteins, including the distantly related TRIO protein. Six genes of this family are known in An. gambiae, five of which reside in chromosome X, while the gene coding for the TRIO protein is in the 2R chromosome arm (supplemental table 4 at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls). Two of these gene products are reported here in their full-length configuration. Four of the five genes in the X chromosome are observed in a tandem configuration, including one in reverse orientation(Fig. 5). This family has a relatively high EST representation in our database, with a total of 114 EST. Except for gSG1a having two EST, all others had 10 or more EST represented (Table 4). One polyadenylated transcript (contig_78) was found mapping in anti-sense orientation of SG1_like-3 (Fig. 5). Its possible significance is unknown. Alignment of the six protein sequences is not very informative, except for a weak similarity region in the middle of the protein (Fig. 6). A hidden Markov model made from the Clustal alignments of the six proteins was used to search the NR protein database of NCBI. All retrieved protein sequences were of anopheline origin (not shown). Evidence for secretion of gSG1b, SG1 and SG1-like3_long was found by Edman degradation of SDS-PAGE protein bands (supplemental table 4 at http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls).

Due to their putative high number of serines and threonines and their high probability of having 10 or more O-linked N-acetylgalactosamine, three proteins are identified as mucins, two of which have been described previously and one of which is novel(Table 4). All of these proteins have homologues found in sialotranscriptomes of An. stephensi, and one in Culex quinquefasciatus. These proteins might function in the lubrication of the mosquito mouthparts. One of them(SG3) has a weak indication of a chitin binding site domain, and it is possible that it binds to the chitinous linings of the salivary ducts and mouthparts. These predicted transcripts were found to be enriched in salivary glands of females and were also found in male mosquitoes(Table 4).

Table 4 lists 33 peptides and proteins with no hits or non-significant e values when compared with GO,PFAM and SMART databases. Fifteen of them were not previously reported as being expressed in the salivary glands of An. gambiae. Additionally,three previously described messages are now reported in their full CDS form. Thirty of these 33 proteins have a signal peptide indicative of secretion,although it should be noted that their final destination could be the ER or Golgi complex. Except for the transcript coding for the protein described before as cE5 (Arca et al.,1999), which is the homologue of the An. albimanusantithrombin peptide named anophelin(Valenzuela et al., 1999), we have no information that could indicate the function of these gene products. Some of these proteins apparently result from gene duplication events, such as those listed in Table 4 as: (1) hyp15 and hyp17, coding for basic peptides (pI>11.0) of∼4.7 kDa and residing contiguously on chromosome X; (2) hyp10 and hyp12, coding for slightly acidic peptides of ∼7.5 kDa residing contiguously on chromosome arm 3R; (3) hyp8.2 and hyp6.2,apparently unrelated in sequence similarity but coding for mature peptides of 7.6 and 6.2 kDa and residing contiguously on chromosome arm 2L; (4) SG2 and SG2A, coding for mature peptides of 9.5 and 15.5 kDa, apparently unrelated in sequence similarity, residing close to each other on 2L and (5) hyp4.2 and hyp13, coding for mature peptides of 4.2 and 3.6 kDa on chromosome arm 2R. These pairs of genes show identical or very similar patterns of expression (see Table 4) and possibly reflect examples of gene duplication and divergence of function(Sankoff, 2001), as do the D7,SG1 and AG5 families described above.

Of these 33 salivary gland-expressed genes, 22 appear to code for proteins found only in anopheline mosquitoes, five are common to Culicidae, one is known to occur also in Drosophila and four are more generally conserved. Together with the six members of the SG1 family, there are 28 gene products that appear to be unique to anophelines and could be used as antigenic markers of anopheline exposure for epidemiologic studies, as done previously for ticks and sand flies (Barral et al., 2000; Schwartz et al.,1990).

Among the gene products unique to mosquitoes (including anophelines and culicines), we report here the full-length information for the 30_kD protein. The 30_kD transcript sequence produced nearly 200 EST matches from our database, being the second most abundantly expressed gene in the salivary glands of An. gambiae. Splice variants of this protein are apparent from the different assemblies of these EST. Transcripts coding for members of this acidic protein family, first identified as the 30-kDa Aedesallergen (Simons and Peng,2001) and also named GE-rich protein(Valenzuela et al., 2003),were found in all previously described transcriptomes of both culicine and anopheline mosquitoes. Another unique mosquito protein family is represented by hyp55.3 (Table 4). Additionally, two An. gambiae genes code for a protein similar to a salivary Culex protein annotated as a putative 14.5-kDa salivary peptide. Although these two Anopheles proteins appear to be related,their corresponding genes are located in different chromosomes. The protein indicated as SG2a also has homology to a Culex putative salivary protein.

One single EST identified an An. gambiae gene coding for a protein with 49% identity to Drosophila retinin, a protein of unknown function expressed in the insect eye. Genes of a more general conserved nature expressed in An. gambiae salivary glands include the previously described selenoprotein, the hypothetical proteins named in Table 4 as hyp14.6 and hyp1.2,and calreticulin. Although calreticulin functions as a chaperone in the ER,and the An. gambiae salivary calreticulin has a carboxy-terminal sequence HDEL suggestive of retention in the ER, proteins of this family have anti-thrombotic functions in the extracellular compartment(Nash et al., 1994; Nauseef et al., 1995; Pike et al., 1998; Sontheimer et al., 1995) and have been described in the saliva of ticks(Jaworski et al., 1995). Its possible function in the saliva of ticks and Anopheles mosquitoes remains to be investigated.

For this broad class of proteins, we found evidence of synthesis of gSG7,gSG7-2 and the 30_kDa peptides by Edman degradation of salivary gland peptides resolved by SDS-PAGE.

Several transcripts coding for enzymes are identifiable. These enzymes are probably associated with four groups of functional activities.

1. Catabolism of hemostasis and inflammation mediators, including the previously described 5′-nucleotidase and apyrase, that may facilitate acquisition of blood meals by removing pharmacologically active nucleotides at the site of the injury (Ribeiro and Francischetti, 2003). We found strong Edman degradation signal in SDS-PAGE bands matching the predicted amino terminal of the putative 5′-nucleotidase. Additionally, we describe a salivary peroxidase homologous to the enzyme in An. albimanus that acts as a vasodilator by its catechol-oxidase activity (Ribeiro and Valenzuela, 1999; Ribeiro and Nussenzveig, 1993). Transcripts for enzymes of this class were well expressed, with 74, 26 and 6 EST found in the database for the 5′-nucleotidase, apyrase and peroxidase, respectively. We also report a gene product coding for an epoxy hydrolase, represented by a single EST match. The corresponding gene, as presently annotated, codes for a truncated protein(ENSANGP00000008689) for which we cannot find the starting methionine and thus infer whether it may be secreted. Notably, this gene is located contiguously and in reverse orientation to the salivary apyrase gene in chromosome arm 3L. It is included in the detailed analysis because of the potential role of arachidonic acid epoxides and epoxide hydrolase in inflammation and hemostasis(Spector et al., 2004; Weintraub et al., 1999).

2. Sugar digestion-related enzymes, including amylase and α-glucosidase,both described in their full-length CDS in this paper.

3. Proteases, including four serine proteases that could be involved with specific host proteolytic events that could affect clotting or the complement cascade. Two other serine proteases are probably involved in immunity, as they are similar to prophenoloxidase-activating enzymes. One metalloprotease homologous to a Drosophila enzyme involved in remodeling of the salivary glands was also found in the salivary transcriptome of An. gambiae. We could not ascertain its full-length coding sequence and therefore the occurrence of a signal peptide, but it is included here for having seven putative O-glycosylation sites and as a reference for future studies regarding its involvement in the development of the adult salivary gland or as a potentially secreted metalloprotease, as occurs with ticks (Francischetti et al.,2003).

4. The homologue of Drosophila peroxinectin is also identified. Peroxinectins are multifunctional proteins with a peroxidase domain and cell adhesion activity. This enzyme may have a role in blood feeding or, more probably, may be involved with sclerotization of extracellular matrix constituents. We found strong Edman degradation signal in SDS-PAGE bands matching the predicted amino terminal of the putative 5′-nucleotidase.

Transcripts coding for two different lysozymes were found, one abundantly transcribed (36 transcripts) and one with only one EST. Lysozyme activity was previously shown to be abundant in salivary glands of both male and female mosquitoes, where it may help contain microbial growth in stored sugar meals(Moreira-Ferro et al., 1998; Rossignol and Lueders, 1986). Two lectin-coding genes were also identified as expressed in the salivary glands: (1) a C-type lectin, found through a match to the 3′-UTR of an mRNA coding for a putative protein with PFAM and SMART domains, indicating this type of lectin, corresponding to ENSANGP00000020547, and (2) galectin,found by one EST match, corresponding to ENSANGP00000026948. Although the encoded An. gambiae galectin does not have a signal peptide indicative of secretion, it is known that galectins may be secreted via an alternative mechanism(Nickel, 2003). If secreted,these proteins may be responsible for the hemaglutinating activity of Anopheles saliva (Metcalf,1945), which can help in concentrating the blood meal(Vaughan et al., 1991).

While the function of most putative salivary proteins and peptides is presently unknown, determination of their tissue and sex specificity may help to direct further research to characterize these gene products. To this end,we used RT–PCR on total RNA extracted from female salivary glands,female carcasses (i.e. adult females from which salivary glands had been removed) and adult males. Eighty-eight mRNA, mostly encoding secreted polypeptides, were selected on the basis of either their sequence similarity or absence of any similarity to known proteins. Nine additional mRNA previously analysed by RT–PCR (AgApy, AgApyL1, D7r1, gSG1, gSG3, gSG6,gSG7, gSG10, cE5/anophelin) were also included as controls for the amplification reactions (Arca et al.,1999; Francischetti et al.,2002; Lanfrancotti et al.,2002). We have also analysed the sex-dependent expression of the genes shown in Table 4 using the Affymetrix microarray chip for comparison with the RT–PCR results. Of the 72 gene products shown in Table 4, three are not represented in the Affymetrix gene set. The combination of these two independent analyses allows the delineation of three categories. The first is represented by genes either expressed at approximately the same level in the three tissues examined or less abundantly transcribed in female glands: proteins encoded by these ubiquitous genes are presumably involved in housekeeping functions. Approximately one-third of the genes analysed (25/72) belong to this group; a few representatives are shown in Fig. 7A. In the microarray experiment, these genes should show equal expression in males and females,and, indeed, the average of the log of the hybridization signal ratio between sugar-fed females and males for this set of genes was 0.11±0.07 (mean± s.e.m.; N=24), a result not significantly different from 0 (a value indicating equal hybridization signal,because 10⁰=1). These ubiquitously expressed genes may play housekeeping roles related to glandular function, such as calreticulin, as well as general immune mechanisms, such as the case of the lyzozymes and prophenoloxidase activating serine proteases.

The second category of tissue-specific expression consists of 34 genes represented by 17 transcripts that are either female salivary gland specific(marked as `SG' in Table 4)(Fig. 7B) or whose expression is enriched in the female salivary glands (the 17 additional genes are marked as `Enrich' in Table 4)(Fig. 7C). Microarray experiments indicated a highly significant differential ratio of expression between sexes in both subgroups. The SG set had a mean log of ratios of 1.45±0.22 (N=17), and the Enrich set had an average ratio of 1.24±0.19 (N=15; two genes are missing on the microarray chip), indicating a geometric average increase of transcript expression of 28-and 17-fold, respectively, when female transcript expression is compared with that of males. Additionally, the hybridization signals for each probe set were analyzed with an algorithm (GCOS) that designated the presence or absence of the corresponding transcript. Again, in most cases, the transcripts identified as female specific by RT–PCR were confirmed by the microarray data.

The female-enriched or female-specific salivary gland genes are likely to play a role in blood feeding as anti-hemostatics or immunomodulators. Nineteen of the 34 genes are either newly described here or had not been previously analyzed for their expression specificity. Jointly, these genes include all eight members of the D7 family, the AG5 protein gVAG, all six members of the SG1 family, the protein/peptides named 30_kDa, gSG7-2, hyp17, gSG7, hyp8.2,gSG6, hyp14.5, gSG5, hyp15, gSG8, hyp37.7, hyp6.2, the enzyme salivary peroxidase, 5′-nucleotidase, apyrase, the serine proteases sal_serpro1,sal_tryp_XII, Sal_serpro2, the salivary epoxy hydrolase and the salivary galectin.

The third subgroup of genes includes those expressed in female salivary glands as well as in adult males, with absent or irrelevant expression in female carcasses (Fig. 7D). Microarray experiments indicated that the average log of the ratio of the hybridization intensities between females and males was not significantly different from 0 (–0.13±0.08; N=12). We assume that these genes are salivary gland specific and expressed both in male and female glands. The corresponding gene products are probably involved in sugar feeding, antimicrobial activity or in more general physiological gland functions. Overall, 13 genes appear to be part of this group, including those encoding three mucins, and the proteins/peptides hyp55.3, hyp10, hyp12, sg2,sg2a, gSG9, hyp6.3, and the sugar-digesting enzymes amylase and maltase(Table 4). With the exception of enzymes that may help sugar digestion in the mosquito crop and midgut, and the mucins that might help maintenance of the food canal, the function of the remaining gene products of this group is presently unknown.

Although the results obtained from the microarray experiment generally agree well with those from the RT–PCR experiments, we found some noticeable discrepancies in the results for the genes sal_galectin,hyp14.5, Sal_serpro2, sal_tryp_XII, Ag_epoxy_hydrolase and AG5-related-4. One of the reasons for the observed incongruity may be related to alternative splicing of the gene products. Indeed, RT–PCR expression analysis followed by cloning and sequencing of the amplified fragments suggested that the salivary galectin (not shown) and sal_tryp XII genes produce different polypeptides by alternative splicing. In the case of female-gland-specific sal_tryp XII, a band 126 bp in length is obtained, as expected, when female salivary gland RNA is used as template,whereas larger products are amplified from RNA extracted from carcasses and males (Fig. 8). Sequence analysis indicated the longer product to be a transcript that retains a 98-bp intron carrying an in-frame stop codon. This would give rise to a hypothetical truncated product of 112 amino acids in place of the putative trypsin-like protease produced in female salivary glands (431 amino acids). It is possible that this tissue- and sex-specific splicing may have a regulatory role,producing a functional protease only in the saliva of An. gambiaefemales. As suggested above, if secreted, it may influence the clotting and/or the complement cascades of vertebrate hosts. Microarray hybridization experiments using a set of 10 or 11 short probes, as used in the Affymetrix chip, cannot distinguish between these splice variants, making such comparisons invalid (Carter et al.,2005). On the other hand, the incongruence between RT–PCR experiments and microarray data may point to differentially spliced genes that may have importance in tissue translation selectivity(Black, 2003).

Two of the nine genes included in this analysis as controls showed an expression pattern slightly different from what has been reported before, most probably because new primer pairs and different amplification conditions were employed. The expression of gSG7 appeared enriched in female glands,rather than equally expressed in female salivary glands and in males, as previously reported (Lanfrancotti et al.,2002), whereas cE5 showed very little expression in carcasses in comparison to what was observed previously(Arca et al., 1999). In Table 4, they have been classified according to the more recent RT–PCR results, although it should be kept in mind that their expression pattern may be in-between these categories. Very good overlap with previous analyses was obtained with the other seven genes used as controls (AgApy, AgApyL1, D7r1, gSG1, gSG3,gSG6 and gSG10). It is also interesting that cE5 is a homologue of anophelin, a potent anti-thrombin peptide found in the salivary glands of the New World mosquito An. albimanus(Francischetti et al., 1999; Valenzuela et al., 1999);however, in An. gambiae, cE5 is not found selectively in female glands, as shown here and previously (Arca et al., 1999), raising the possibility that it may exert a different function in Old World mosquitoes.

Using high-throughput transcriptome analysis, we significantly expanded the An. gambiae salivary gland transcript repertoire. Thirty-three novel putative salivary proteins were identified, and the full-length sequences of seven previously identified partial cDNA were reported. Moreover,tissue-specific expression studies on selected clones allowed us to identify 27 additional genes that are either enriched or specifically expressed in the salivary glands. The information obtained in the course of this analysis,combined with the results from previous studies, allowed us to compile an updated catalogue that includes a total of 72 transcripts, mainly encoding putative secreted products. Forty-seven of these transcripts encode proteins that may play essential physiological roles, as indicated by their exclusive or preferential expression in female and/or male salivary glands. This catalogue makes the mosquito An. gambiae the arthropod disease vector for which the most complete salivary transcriptome is available. On the other hand, the fraction of genes included in this list for which we know or can postulate a function is surprisingly small, emphasizing how much we still have to learn about bioactive molecules from the saliva of blood-feeding arthropods. We believe that this updated catalogue should help our continuing effort of understanding the evolution of blood sucking in vector arthropods and the discovery of novel pharmacologically active compounds.

We are grateful to Brenda Marshal for editorial assistance.

This work was supported in part by grants from the European Union to M.C. and B.A. (BioMalPar N 503578), MIUR/COFIN funds to Vincenzo Petrarca and B.A.,the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health to J.M.C.R., and by the UND/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), ID A20314 to O.M.