Figure 1.
Mass spectral data for purified recombinant CquiOBP1.
(A) HPLC separation, (B) mass spectrum of CquiOBP1 peak, and (C) deconvolution data indicating a molecular mass of 14,479 Da.
Figure 2.
Gel filtration elution profiles of CquiOBP1.
(A) Monomeric and (B) dimeric form of CquiOBP1. The dimer was isolated with a minor peak of the monomer. The dimer dissociates into monomer as indicated by the increase in the second peak (C) after 1 hour at room temperature and (D) overnight at 4°C. The dimeric form is also dissociated with organic solvent. (E) Sample D analyzed with acetonitrile in the mobile phase.
Figure 3.
Oviposition Pheromone Reception.
(A) Single sensillum recordings from short, sharp-tipped trichoid sensilla on the antennae of female Cx. quinquefasciatus. Response of a neuron to both the natural stereoisomer, (5R,6S)-MOP, and its antipode. The sensilla housed a second olfactory receptor neuron, characterized by a smaller spike amplitude, which was very sensitive to skatole. Bar denotes stimulus duration, 500 ms. Immunohistochemical localization of CquiOBP1 in the trichoid (B) long, sharp-tipped sensilla, (C) long, sharp-tipped sensilla with high density labeling at the excised tip, (D) MOP-detecting short, sharp-tipped sensilla, and (E) blunt-tipped sensilla on the antennae of female Cx. quinquefasciatus. CquiOBP1 was not detected in the (F) grooved peg sensilla on the antennae, and (G) the peg sensilla on maxillary palps. Scale bars, B, C: 10 µm, others, 5 µm. (H) Western blot analysis of protein extracted from olfactory tissues compared to recombinant CquiOBP1. (I) Same analysis as in H, but with 5× lower amounts of rCquiOBP1 and antennal extract. ANT, antenna-equivalent; MP, maxillary palp-equivalent. While signal was detected from 10 antenna-equivalent, no signal was observed from extracts of 100 maxillary palp-equivalent.
Figure 4.
Binding of test ligands to antennae-specific CquiOBP1.
(A) pH-dependent binding of racemic MOP to CquiOBP1. (B) Binding of enantiomers compared to racemic MOP. The non-natural stereoisomer, (5S,6R)-MOP showed significantly higher affinity for CquiOBP1 than the natural pheromone. (C) Nonanal bound to CquiOBP1 with high affinity at high but not low pH, whereas 1-octen-3-ol did not bind the protein at high or low pH.
Figure 5.
Competitive binding of MOP to CquiOBP1.
Fluorescence emission spectra of CquiOBP1 alone (15 µg/ml; black), in the presence of NPN (2 µl, 3.2 mM; light red), and after titrating with increasing amounts of MOP (1–3 µl, 3.2 mM; green, blue, and dark red lines). (A) Replacement of fluorescent reporter by MOP is indicated by decrease of emission, and suggests competitive binding. (B) Excitation of the fluorescent reporter was not changed with addition of MOP thus indicating no MOP binding at low pH.
Figure 6.
Far-UV circular dichroism spectra of CquiOBP1 at pH 6.5 (blue) and pH 5 (green).
The helical-rich protein underwent unwinding of α-helix at low pH as indicated by the change in the intensity of the second minima.
Figure 7.
Resolution of MOP stereoisomers on a chiral column.
(A) Partially resolved enantiomers of MOP. (B) Random mixture of stereoisomers showing two clusters of well separated stereoisomers: erythro- and threo-MOP. (C) The pheromone isolated from egg rafts of Cx. quinquefasciatus showed the same retention time as the second peak in the erythro-MOP cluster, and is thus confirmed to be (5R,6S)-MOP. (D) The natural stereochemistry and (E) the configuration of the antipode were retained upon binding. (F) Competitive binding with the two enantiomers showed that CquiOBP1 has a higher affinity for the non-natural stereoisomer (first peak) than for natural stereoisomer, (5R,6S)-MOP (the second peak).
Figure 8.
GC-EAD analysis of rabbit chow fermentation products.
The three EAD-active peaks (red arrows) were identified as (1) trimethylamine, (2) nonanal, and (3) skatole.
Figure 9.
Field data comparing captures of female Cx. quinquefasciatus in synthetic mixtures- and infusion-baited traps.
Catches in traps baited with nonanal and TMA, skatole and TMA, and infusion were not significantly different (Tukey HSD, P>0.05), but the nonanal plus TMA lure is odorless and thus suitable for surveillance and use in monitoring population in human dwellings.
Figure 10.
Catches in traps baited with a synthetic attractant alone or in combination with MOP.
Captures in traps loaded with pheromone were not significantly different (Tukey HSD, N = 15, P>0.05) from those in trap baited only with nonanal plus TMA.