1. Aedes aegypti mosquitoes can sense bitter substances but are not repelled
by bitters applied to human arms
Vineeta T. Reddy, Emily J. Dennis, and Leslie B. Vosshall
Laboratory of Neurogenetics and Behavior, Howard Hughes Medical Institute, The Rockefeller University, New York, NY
INTRODUCTION:
ABSTRACT:
METHODS :
RESULTS: CONCLUSIONS:
REFERENCES:
1 Lee, Y., Kim, S. H. & Montell, C. Avoiding DEET through Insect Gustatory
Receptors. Neuron 67, 555–561 (2010).
2 Matthew DeGennaro et al. orco mutant mosquitoes lose strong preference
for humans and are not repelled by volatile DEET. Nature 498, 487-491
(2013).
3 Sanford, J. L., Shields, V. D. C. & Dickens, J. C. Gustatory receptor neuron
responds to DEET and other insect repellents in the yellow-fever mosquito,
Aedes aegypti. Naturwissenschaften 100, 269–273 (2013).
ACKNOWLEDGEMENTS:
10% sucrose + solvent
10% sucrose
+ 1% DEET
or bitters, or
control (solvent)
Thanks to everyone in the Vosshall lab for the constant
support and guidance, and a special thanks to my lab
head, Leslie B. Vosshall, for hosting me in the lab, and
my mentor, Emily J. Dennis, for taking me on and
helping me every step of the way.
FUTURE DIRECTIONS:
DEET (N,N-Diethyl-meta-toluamide) is the most common
and broadly effective insect repellent available. Even though
we have used DEET for over sixty years, its mechanism of
action remains elusive. Behaviorally, DEET repels
mosquitoes at a distance on olfactory receptor neurons, and
requires the olfactory co-receptor orco. Aedes aegypti orco
mutants will land on DEET-treated arms, but do not blood-
feed, sensing DEET through unknown chemosensory
receptor(s).
There are gustatory receptor neurons in the proboscis of
Aedes aegypti mosquitoes that respond to both DEET and
bitter compounds, indicating a possible mechanism for
contact DEET repellency. We predict that if these neurons are
responsible for contact DEET chemorepellency, then bitter
substances should mimic the effects of DEET. We used a two-
choice, capillary feeder (café) assay to study the effects of
DEET and bitters on sugar feeding, and to establish
behaviorally relevant concentrations of these compounds. We
then tested the concentrations of these bitter compounds on
human arms to see if they could mimic DEET.
We determined that mosquitoes avoid DEET and bitter
solutions in the café assay, but bitter compounds cannot
mimic DEET when applied to human arms. This indicates
that there are other neurons involved in the orco-independent
rejection of DEET-treated arms.
DEET is a synthetic chemical not found in nature. It was
discovered as a part of a large chemical screen in the late
1940s, and has since become the most effective and widely
used arthropod repellent available. Even though it has been
in wide public use since the 1950s, the molecular
mechanisms of DEET repellency remain elusive.
Previously, it was difficult to study close-range effects of
DEET because of its long-range effects: mosquitoes are
repelled by DEET at a distance, and rarely land on the skin,
where contact-mediated DEET repellency occurs. Using
recently developed orco mutant mosquitoes that cannot
smell DEET, we can now begin to disentangle the
multisensory effects of DEET. These orco mutants will land
on DEET-treated arms but will not blood-feed on them.
Café assay
Mosquitoes were:
-starved for 48 hours
-placed in a vial and given a
choice between a capillary
filled with a sucrose and
solvent mixture and a second
capillary containing a mixture
of sucrose and DEET or bitters
in solvent
-after 4 hours, we measured the
amount of solution left in each
capillary
-to control for the effects of
evaporation, we subtracted the
average amount of sugar
missing from capillaries placed
in empty vials
Mosquitoes were:
-starved overnight in
groups of 25 mosquitoes
-put into a cage for five
minutes to acclimate
-tested for ten minutes
on an arm treated with
900uL
solvent, 1%
DEET, or bitters
-scored for blood-
feeding
Biting assay
• Wild type and orco mutant mosquitoes avoid a sugar
solution containing DEET or bitters, confirming that
this is an orco-independent phenotype.
• Bitter substances cannot mimic the effects of DEET
when applied to human skin, indicating there are
likely undiscovered neurons necessary for contact
DEET repellency.
• Test higher concentrations of bitters in the café assay
with Aedes aegypti to see if they evoke a stronger
phenotype. If they do, test those concentrations on the
arm to see if the response can mimic DEET.
• Study Drosophila melanogaster in the café assay to
try and separate the role of the proboscis and the legs
in sugar preference and DEET avoidance.
• Use an artificial blood feeder to model the effects of
DEET on blood feeding.
Recently, researchers identified a neuron in the labellum of
Aedes aegypti mosquitoes that respond to both bitters and
DEET. 3 We hypothesized that these neurons could be
responsible for the effects of DEET on contact in Aedes
aegypti. This hypothesis predicts that bitters should be able
to mimic the effects of DEET. Long-term, determining
which neurons are behaviorally relevant for DEET
repellency could contribute to the intelligent design of next-
generation insect repellents around the world.
Contact-mediated DEET repellency on human arms
cannot be mimicked by bitters
Behavioral avoidance of DEET in sugar feeding
can be mimicked by bitters
solvent
DEET
1
0
-1
*** ***
Preferenceindex
(uLsucroseconsumed-uLDEET
solutionconsumed)/totalconsumed
+/+ orco5/16
orco16/+
10% sucrose
+ 1% DEET
or solvent
10% sucrose
+ solvent
1
-1
0
solvent
1mM lobeline
*
10% sucrose
+ bitter
or solvent
10% sucrose
+ solvent
Preferenceindex
1
-1
0
**
solvent
5mM quinine
all orco5/16
BloodFedMosquitoes(%)
100
50
0
Solvent
(Ethanol)
Quinine
5mM
Lobeline
5mM
1% DEET
52mM
1
-1
0
solvent
1mM lobeline
*
10% sucrose
+ bitter
or solvent
10% sucrose
+ solvent
Preferenceindex
1
-1
0
**
solvent
5mM quinine
all orco5/16
solvent
DEET
1
0
-1
*** ***
Preferenceindex
(uLsucroseconsumed-uLDEET
solutionconsumed)/totalconsumed
+/+ orco5/16
orco16/+
10% sucrose
+ 1% DEET
or solvent
10% sucrose
+ solvent