MOSQUITO BIOLOGY
Mosquito heat seeking is driven by an ancestral
cooling receptor
Chloe Greppi^1 , Willem J. Laursen^1 , Gonzalo Budelli^1 , Elaine C. Chang^1 , Abigail M. Daniels^1 ,
Lena van Giesen^1 , Andrea L. Smidler2,3, Flaminia Catteruccia^2 , Paul A. Garrity^1 †
Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to
locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. Here,
we identify ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator
of heat seeking in the malaria vectorAnopheles gambiae. AlthoughIr21amediates heat avoidance in
Drosophila, we find it drives heat seeking and heat-stimulated blood feeding inAnopheles. At a cellular
level,Ir21ais essential for the detection of cooling, suggesting that during evolution mosquito heat
seeking relied on cooling-mediated repulsion. Our data indicate that the evolution of blood feeding in
Anophelesinvolves repurposing an ancestral thermoreceptor from non–blood-feeding Diptera.
I
nsect-borne diseases kill over 700,000
people annually, with >400,000 deaths
resulting from malaria, a disease caused
by protozoanPlasmodiumspp. parasites
that are transmitted by blood-feeding
anopheline mosquitoes ( 1 ). Host seeking by
mosquitoes and other pathogen-spreading in-
sects relies on the detection of host-associated
cues, including carbon dioxide (CO 2 ), odors,
and body heat ( 2 – 5 ). Receptors for CO 2 and host
odors have been characterized in mosquitoes
( 6 – 9 ), but receptors that promote heat seek-
ing and heat-induced blood feeding have re-
mained elusive ( 4 , 10 – 12 ). As vector mosquitoes
are descendants of non–blood-feeding ances-
tors ( 13 ), it remains unknown whether the
emergence of heat seeking and warming-
induced blood feeding inmosquitoes involved
the generation of novel thermoreceptors or
the repurposing of existing thermoreceptors.
To date, mosquito orthologs of twoDrosophila
warmth receptors, TRPA1 ( 14 )andGR28b( 15 ),
have been tested as candidate heat-seeking
receptors in the yellow fever mosquitoAedes
aegypti( 10 – 12 ). However, neither is required
for heat seeking inAedes( 12 ). Rather, TRPA1
promotes heat avoidance in bothAedesand
Drosophila( 12 , 14 ). Although efforts have fo-
cused on warmth receptors, insects also pos-
sess cooling-activated receptors, which should
be equally capable of supporting heat seek-
ing through cooling-mediated repulsion. In
Drosophila,cooling detection is mediated by
IR21a, IR25a, and IR93a ( 16 – 18 ), three members
of the ionotropic receptor (IR) family, a group of
invertebrate-specific sensory receptors related
to ionotropic glutamate receptors ( 19 ). IR21a is
specifically required for cooling detection in
the fly and can confer cooling sensitivity when
ectopically expressed ( 16 , 18 ), while IR25a and
IR93a are more broadly acting co-receptors
that support cooling detection and other IR-
dependent sensory modalities ( 17 , 19 , 20 ). At
the behavioral level inDrosophila, IR21a,
IR25a, and IR93a help the fly achieve optimal
body temperatures by supporting avoidance
of excessively cool and warm temperatures
( 16 , 18 ). BeyondDrosophila, IR21a, Ir25a, and
IR93a are each widely conserved from Diptera
(flies and mosquitoes) to Isoptera (termites)
( 19 ), raisingthe possibility that their ther-
mosensory functions may also be conserved.
UsingAnopheles gambiae, a major vector of
malaria in sub-Saharan Africa, we first tested
whether IR21a is required for detecting cool-
ing in mosquitoes and subsequently whether it
can drive heat attraction and heat-stimulated
blood feeding.
Two mutant alleles ofA. gambiae Ir21awere
generated using CRISPR-Cas9 (see methods).
Ir21a+7bpcontains a 7-base pair (bp) insertion,
introducing a frameshift positioned to dis-
rupt IR21a’s translation within the second of
IR21a’s three transmembrane domains; this
lesion is predicted to generate a nonfunctional
receptor (Fig. 1A). InIr21aEYFP, a disruption
cassette containing an enhanced yellow fluo-
rescent protein (EYFP) marker, was inserted
into IR21a’s fourth exon, a lesion also pre-
dicted to create a nonfunctional receptor (Fig.
1B). Both mutants lacked detectable IR21a
protein expression (Fig. 1, C to E, and fig. S1),
consistent with their acting asIr21anull
mutations.
Genome-wide analyses ofA. gambiaesen-
sory tissues suggest thatIr21aRNA is specif-
ically expressed in the antenna ( 21 ). To visualize
IR21a protein expression and localization
with cellular resolution, anti-IR21a antisera
were generated. The antenna’s most distal seg-
ment (flagellomere 13) contains three coeloconic
sensilla that house sensitive thermoreceptors
( 22 – 24 ) (Fig. 1C). In females, IR21a expressionwas detected in three sensory neurons in fla-
gellomere 13, one innervating each of the co-
eloconic sensilla (Fig. 1D). Consistent with a
role in thermosensory transduction, IR21a
strongly localized to the sensory ending of each
of these neurons (Fig. 1D). IR21a immunostain-
ing was absent inIr21amutants, confirming
antisera specificity (Fig. 1E and fig. S1A). The
male antennal tip also contains thermorecep-
tors ( 23 ), and IR21a expression was detected
in sensory endings there as well (fig. S1B).
Extracellular recordings were performed
from the IR21a-positive coeloconic sensilla at
the antennal tip (Fig. 2A). In wild-type mos-
quitoes, the activity of the Cooling Cell, a ther-
mosensory neuron stimulated by cooling and
inhibited by warming, was readily detected
(Fig. 2B). On rare occasions of exceptional
signal to noise, a smaller-amplitude spike was
also detected, corresponding to a Heating Cell
activated by warming and inhibited by cool-
ing (fig. S2). Cooling Cell responses were
highly thermosensitive: an ~0.5°C drop from
~30°C increased spiking by ~40%, and an
~0.5°C drop from ~37°C increased spiking
by ~80% (Fig. 2C). Response adaptation ini-
tiated rapidly, followed by a slower decline to
baseline (Fig. 2C and fig. S3). Heating inhib-
ited spiking, in a similarly transient manner
(Fig. 2C). Importantly, Cooling Cells remained
highly active at warm temperatures (e.g., 37°C)
and were neither more active nor more thermo-
sensitive at colder temperatures (Fig. 2, C and
D). Thus, while often referred to as Cold Cells
in the classical literature, cooling and not
cold is their activating stimulus. In addi-
tion, while often referred to as“phasic-tonic”
receptors, their responses to temperature shifts
adapted fully, albeit slowly, requiring sus-
tained observation (>20 s) to fully appreciate
(fig. S3). Therefore, although they fire robust-
ly at constant temperature, Cooling Cells are
phasic thermoreceptors.Their rate of baseline
firing was relatively temperature insensitive
with a fold change upon 10°C increase [Q10 of
~1.6, reflecting a slight increase with warmth],
enabling the cell to respond to small tempera-
ture fluctuations over a wide range of absolute
temperatures. [Physiologically similar Cooling
and Heating Cells have been described in
A. aegypti( 22 , 24 )andDrosophila melanogaster
( 18 ).] Taken together, these data indicate that
Cooling Cells are phasic thermoreceptors that
respond to temperature change rather than ab-
solute temperature and that they are capable
of responding to abrupt changes in tempera-
ture over the wide range of absolute tempera-
tures relevant for host seeking (Fig. 2, C and D).
Cooling Cell thermosensitivity was elimi-
nated inA. gambiae Ir21amutants. The large-
amplitude spike detected inIr21amutants
was neither activated by cooling nor inhibited
by warming (Fig. 2, B to D). Rather, its activity
increased slightly upon warming, with a Q10RESEARCH
Greppiet al.,Science 367 , 681–684 (2020) 7 February 2020 1of4
(^1) Department of Biology and Volen Center for Complex
Systems, Brandeis University, Waltham, MA 02453, USA.
(^2) Department of Immunology and Infectious Diseases,
Harvard T. H. Chan School of Public Health, Boston, MA
02115, USA.^3 Wyss Institute for Biologically Inspired
Engineering, Boston, MA 02115, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected]