(fig.S4,HandI)mosquitoes,thispreferencefor
warm blood was significantly reduced. Thus,
similar to heat seeking, warmth-promoted blood
feeding was reduced in the absence ofIr21a.
ThesedataidentifyIR21aasakeymediator
of heat-seeking behavior inA. gambiaemos-
quitoes. Although a cooling-activated receptor
driving heat seeking is superficially counter-
intuitive, repulsion from cooling would yield a
similar behavioral outcome as attraction to
warming. Furthermore, Cooling Cells are bidi-
rectional and are not only activated by cooling
but also inhibited by heating (Fig. 2, D and E);
each phase of the response could modulate
downstream circuits to control behavior. Ulti-
mately, the detection of temperature change
by the Cooling Cells is critical, but is just one
step in heat seeking, a response that involves
the processing of multiple sensory inputs to
generate a coherent response. Identification
of a key molecular receptor for heat seeking
provides a starting point for a deeper under-
standing of this complex behavior and its
contribution to the multimodal process that
culminates in mosquito blood feeding.
The conservation of IR21a’sthermosensory
function betweenDrosophila( 18 )andAnopheles
(Fig. 2), whose last common ancestor lived
~250 million years ago, suggests thermosens-
ing is an ancestral function of IR21a. As this
ancestor predates the evolution of blood feed-
ing ( 13 ), its IR21a would have regulated other
behaviors, such as thermoregulation. Thus,
our findings indicate that the evolution of
blood feeding inA. gambiaemosquitoes in-
volved repurposing an ancestral thermo-
receptor to facilitate host seeking. Alterations
in the connectivity or function of downstream
circuits would likely have been crucial in this
behavioral shift. Given the conservation of
IR21a as well as IR25a and IR93a (IR21a’sco-
receptors inDrosophila) across insects ( 19 ),
these IRs may be used in heat seeking not
only by other mosquitoes but also across a
range of hematophagous insect taxa.
In addition toIr21a’s role in heat seeking,
IR21aexpressionintheantennaeofA. gambiae
males suggests it continues to serve addi-
tional thermosensory functions. It will be in-
teresting to assess whether IR21a mediates
thermal preference in male and possibly fe-
male mosquitoes and the extent to which
thermal preference andheat-seeking circuits
overlap. Not all thermoreceptors appear to
have been repurposed, as the TRPA1 warmth
receptor has a similar role in flies and mos-
quitoes, mediating heat avoidance in both ( 12 ).
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ACKNOWLEDGMENTS
We thank A. Hammond, R. Harrell, T. Nolan, S. McIver, A. Crisanti,
E. Marois, B. White, and L. Vosshall for reagents and advice,
R. Albuquerque for assistance with data analysis, R. Gerber for
assistance with mosquito husbandry and behavioral assays,
and A. Lee, E. Marder, M. Rosbash, P. Sengupta, and C. Zhu for
comments on the manuscript.Funding:This work was supported
by grants from the National Institute of Allergy and Infectious
Diseases (F31 AI133945 to C.G.; R01 AI122802 and R21 AI140018
to P.A.G. and F.C.), the National Institute of Neurological Disorders
and Stroke (2T32NS007292-31) to W.J.L., the National Institute
of General Medicine (F32 GM113318) to B.G., the Swiss National
Science Foundation (P2FRP3_168480) to L.V.G., a Faculty Research
Scholar Award by the Howard Hughes Medical Institute and the
Bill & Melinda Gates Foundation (grant OPP1158190) to F.C.,
and the National Science Foundation (IOS 1557781) to P.A.G.
Author contributions:C.G., W.J.L., G.B., L.V.G., A.L.S., F.C., and
P.A.G. designed experiments. C.G., W.J.L., E.C.C., A.M.D., and L.V.G.
performed husbandry, molecular genetics, and heat-seeking
behavior experiments. C.G. performed NHEJ-based gene
disruption. W.J.L. performed gene targeting. W.J.L. and E.C.C.
performed hand approach and blood-feeding behavior analyses.
L.V.G. and C.G. performed immunohistochemistry. G.B. and C.G.
performed electrophysiology. A.L.S. performed transgenesis of
gRNA expression vector. W.J.L. and P.A.G. performed data analysis.
C.G., W.J.L., and P.A.G. wrote the paper with input from all
authors.Competing interests:A.L.S. is a coinventor on patent
WO2015105928A1 (WIPO PCT pending; inventors K. Esvelt and
A. L. Smidler),“RNA-guided gene drives.”The patent involves
spreading desirable traits genetically through mosquito populations
using Cas9-based gene drives. IR21a could potentially be used
as a target for such a gene drive. P.A.G. is a coinventor on patent
WO2017196861A1 (WIPO PCT pending; inventors Z. Knecht,
P.Garrity, L. Ni)“Methods for modulating insect hygro- and/or
thermosensation.”This patent proposes using members of
the ionotropic receptor family as targets for strategies to disrupt
hygro- and thermo-sensation in insects.Data and materials
availability:The datasets generated and analyzed during the
current study are available at DRYAD (https://doi.org/10.5061/
dryad.pzgmsbcg3).SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/367/6478/681/suppl/DC1
Materials and Methods
Figs. S1 to S5
References ( 28 – 32 )
Movies S1 and S2
View/request a protocol for this paper fromBio-protocol.4 August 2019; accepted 6 December 2019
10.1126/science.aay9847Greppiet al.,Science 367 , 681–684 (2020) 7 February 2020 4of4
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