e.m., n = 10). In a separate study, post-mated females were kept at 26 °C for different time periods (0.5 h and 2 h) before the reproductive tissues were removed for extraction and analysis by MALDI/TOF-MS. Aea-HP-1 was detected in tissues from all 0.5 h post-mated females (n = 15, but only 1 out of 10 samples for the 2 h post-mated
females. We used confocal microscopy to determine the volume of a single MAG as 1.67 ± 0.08 nl (mean ± s.e.m., n = 4), AZD8055 order which allowed us to estimate the Aea-HP-1 concentration in the MAGs to be around 400 μM. Reproductive tissues of A. aegypti are known to be rich in peptidases that might be involved in the metabolism of MAG peptides . We confirmed the presence of peptide-degrading peptidases using the insect peptide, APSGFLGVRamide, as a substrate. Under conditions that resulted in over 96% hydrolysis of APSGFLGVRamide, only 8% of Aea-HP-1 was degraded, BI6727 demonstrating the relative stability of Aea-HP-1 to MAG enzymes ( Fig. 5). The most studied peptide of insect MAGs is the sex peptide (SP) of D. melanogaster. This 36 amino acid peptide has not been found outside of a
sub-group of closely related Drosophilidae. It has multiple signaling roles in the post-mated female, the best known of which is a decrease in sexual receptivity to courting males. Recently, it has been shown that SP and insect myoinhibitory peptides (MIPs) are ligands for the same G-protein coupled receptor despite lack of structural similarity; MIPs, like Aea-HP-1, are relatively short peptides (generally 9–12 amino acids) with an amidated C-terminus. This promiscuity of the SP/MIP receptor led us to test whether Aea-HP-1 might be an additional agonist for this receptor. We therefore carried out experiments to see if Aea-HP-1 could elicit a post-mating response in virgin female D. melanogaster ( Fig. 6) . We also tested directly whether Aea-HP-1 was an agonist of the SP/MIP receptor of either D. melanogaster or A. aegypti using an established cell-based assay for receptor activation ( Fig. 7) . Aea-HP-1
did not elicit rejection of male advances when injected into the hemocoel of virgin D. melanogaster females and did not activate the SP/MIP receptors AMP deaminase up to 10 μM. We have for the first time chemically characterized a peptide (Aea-HP-1) with biological activity from the MAG of a mosquito and shown that this molecule is transferred to the female on copulation. Aea-HP-1 is a ten amino acid peptide that was first isolated from >600,000 heads of mixed-sex mosquitoes in 1989 together with the tripeptide Aea-HP-2 (TRFamide) using a radioimmunoassay for the molluscan peptide FMRFamide to guide purification . Aea-HP-3 and a pentapeptide C-terminal fragment (Aea-HP-4) were subsequently found in extracts of the abdomen of adult A. aegypti in addition to Aea-HP-1 .