Location and structure of the salivary glands in the fourth-instar larva of An. gambiae. A) Micrograph showing the location of the salivary glands in relation to the digestive tract. Exoskeleton, fat body and other tissues were previously removed. B) External structure of the salivary gland’s lobes and the salivary duct (only one gland shown). C) Internal structure of the salivary glands. The lumen of both lobes can be visualized in this laser confocal micrograph of a paraffin section labeled with FITC-conjugated Erythrina cristagalli lectin, which displays high binding affinity for the salivary gland tissue (shown in bright green). AL, anterior lobe. AMG, anterior midgut. GC, gastric caeca. HC, head capsule. L, lumen. MT, Malpighian tubes. PL, posterior lobe. PMG, posterior midgut. SD, salivary duct. SG, salivary gland.

Relative abundance of transcripts encoding putative secreted, non-secreted and unknown proteins in the larval SG of An. gambiae, as suggested by microarray analysis. Transcripts encoding secreted proteins were 2.8 times more abundant than those encoding non-secreted proteins. Bar height represents the mean enrichment of transcripts within each functional group. Error bars show the standard error of the mean.

Nucleotide and deduced amino acid sequence of AgDef5. The first 23 amino acids (underlined) form a signal sequence, suggesting a secreted protein. The asterisk indicates the position of the stop codon.

Histochemical detection of mucins in the larval SG. Confocal micrograph showing a section of the thorax of a 4^th instar An. gambiae larva stained with fluorescently-labeled wheat-germ agglutinin. Clear staining can be seen in the anterior lobe of both salivary glands (lumen), as well as in the food bolus, caecal membranes and perithtophic membrane. AL, anterior lobe of salivary gland. CM, caecal membrane. E, exosqueleton. FB, food bolus. H, head. L, lumen of salivary gland’s anterior lobe. PL, posterior lobe of salivary gland. PM, peritrophic matrix.

Two-dimensional gel electrophoresis of larval SG. Bands identified to a protein by triptic digestion / MS are shown. Numbers on the left correspond to molecular weight marker bands shown in the gel. The positive (+) and negative (−) signs below the gel mark the polarity of the isoelectrofocusing dimension. AcCoAD, acyl-coenzyme A dehydrogenase. ATPS-a, ATP synthase alpha subunit. ATPS-b, ATP synthase beta subunit. HSP-70, heat shock protein 70. PDI-1, protein disulfide isomerase 1. PDI-2, protein disulfide isomerase 2. RACK1, receptor for activated protein kinase c. ENSEMBL protein identifiers associated with each band are listed in the supplementary materials, table 3.

Validation of microarray data. A linear regression model (equation and dashed line) revealed a high degree of correlation (R²>0.89) between results obtained by microarray analysis (x-axis) and qRT-PCR analysis (y-axis).

Relative abundance of transcripts associated with putative secreted proteins, grouped by functional annotation. Transcripts associated with immune functions were the most abundant, followed by lipocalins and digestive/detoxifying enzymes. Bar height represents the mean enrichment of transcripts within each functional group. Error bars show the standard error of the mean.

Comparison of the amino acid sequence of AgDef5 with other known dipteran defensins. A) The predicted mature peptide sequence of Anopheles gambiae AgDef5 is aligned with those of Anopheles gambiae AgDef1-4 (ENSEMBL accessions AGAP011294-PA, AGAP004632-PA, AGAP007199-PA, AGAP005416-PA, respectively), Aedes aegypti AeDefA-C (ENSEMBL accessions AAEL003841-PA, AAEL003857-PA, AAEL003832-PA, respectively), and Drosophila melanogaster DmDef (ENSEMBL accession FBgn0010385). Conserved residues are shown in grey background; the peptide sequence of AgDef5 contains the six conserved cystein residues characteristic of all insect defensins. Corresponding cysteins for disulfide bridge formation are connected with dashed lines. Homology annotation of these residues was performed using the known structure of sapecin from the flesh fly Sarcophaga peregrina (GenBank accession number P18313) as a model. B) Neighbor-joining phylogenetic analysis of dipteran defensin sequences using 100,000 bootstrap replicates and pairwise comparison. AgDef5 is placed in the cluster of highly divergent defensins (HDD) formed by AgDef3, AgDef4 and AgDef2 (Christophides et al., 2002). Numbers in the phylogram nodes indicate the percent bootstrap support. Overall tree topology was further confirmed using maximum parsimony analysis (data not shown).

Relative abundance of transcripts associated with putative non-secreted proteins, grouped by functional annotation. Bar height represents the mean enrichment of transcripts within each functional group. Error bars show the standard error of the mean.