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.