The highest concentration of VUAA1 to which the PP-Orco cells were exposed was 2 mM and the assay buffer that was used to evaluate this condition was prepared by injecting roughly 1 µL of a 400 mM stock solution of the molecule in DMSO into 200 µL of Hank’s buffer. Since DMSO is known to impact cells in myriad ways and could potentially interfere with the activity of Orco, we also assessed the behavior of PP-Orco cells in Hank’s buffer that was injected with 1 µL of pure DMSO. This volume is nearly identical to volume of DMSO used in the 2 mM VUAA1 test, and we monitored the cells in buffers containing 1 mM and 5 mM Ca2+ (Figure 7 ).
Figure 7: DMSO does not interact with Orco and can be used to solubilize hydrophobic odorants in the assay buffer.
The normalized fluorescence emitted by the PP-Orco cultures is slightly higher than the signal produced by cultures of P. pastoris GS115 but this difference is not statistically significant. Moreover, the trend that is observed does not mirror that of PP-Orco cultures exposed to VUAA1. There is also no significant difference between the normalized fluorescence emitted by PP-Orco in media containing 1 mM and 5 mM of extracellular calcium. These observations, in conjunction with data recorded in the subsequent experiment using citronella oil, verify that DMSO does not impact Orco in any capacity at the volumes that were considered in this study.
3.6. A component in citronella oil could be modulating Orco
Citronella oil is an essential oil that is extracted from the leaves and stem of lemongrass and is widely used around the world as an insect repellant [12]. The active repellatory agent in citronella oil is the monoterpenoid molecule, citronellal. While the interaction between citronellal and Orco has been studied previously in D. melanogaster [46], its effect on Orco in A. gambiae is unclear. We employed the PP-Orco biosensor to systematically probe the interaction between citronella oil and Orco in Hank’s buffer containing 5 mM of Ca2+. Since citronella oil is highly volatile and largely insoluble in aqueous solutions, which precludes accurate molarity measurements, we pipetted 1 µL of 1:1, 1:3, 1:7 and 1:15 mixtures (volume basis) of citronella oil and DMSO directly into the assay buffer in each well and recorded the fluorescence over 6 minutes. The citronella oil comprises 93% citronellal. We did not record a statistically significant difference between the fluorescence emissions by the samples (Figure 8 ). However, the addition of VUAA1 to a final concentration of 1 mM in the medium approximately 2 minutes into the run activated Orco and generated a TRP-like response. Importantly, the activation was proportional to how diluted citronellal oil was in the solution. Additionally, reversing the order of addition of VUAA1 and citronella oil did not dampen the fluorescence. These observations suggest that citronellal or another constituent of citronella oil is either a negative allosteric modulator of Orco or an antagonist that is weakly competitive with VUAA1. We did not investigate this phenomenon further but it warrants a detailed examination in a future study.