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under 2, which is average for a biological pro-
cess. Thus,Ir21ais essential for thermosensing
by Cooling Cells in the mosquito, demonstrat-
ing thatA. gambiaeIR21a’smolecularfunction
is conserved with itsDrosophilaortholog ( 18 ).
In female mosquitoes, heat seeking is part of
a multimodal host-seeking program activated
upon exposure to CO 2 ,withbodyheatserving
as an important cue close to the host (within
~10 to 15 cm) ( 3 , 8 , 12 ). To assess heat seeking,
female mosquitoes were provided a 20-s puff
of 4% CO 2 and exposed to two targets, a con-
trol target at ambient temperature (~26°C)
and a heated target at ~37°C (Fig. 3, A and B,

and fig. S4A). Wild-type mosquitoes exhibited

robust heat seeking, with 43 ± 3% of CO 2 -

activated mosquitoes landing on the 37°C tar-

get (average ± SEM) (Fig. 3, C and F; movie S1;

and fig. S4B). The loss ofIr21agreatly reduced

this behavior, with only 15 ± 4% ofIr21aEYFP

mutants and 14 ± 4% ofIr21a+7bpmutants

landing on the 37°C target (Fig. 3, D to F;

movie S2; and fig. S4B). In all cases, the con-

trol target was largely ignored, confirming

temperature’simportanceintheassay(Fig.3,

C to F). While heat seeking was greatly re-

duced inIr21amutants, it was not entirely

eliminated (Fig. 3, D to F). This residual activ-

ity likely reflects signaling from other as-yet-

uncharacterized thermosensors. However, the

strong reduction of heat seeking inIr21amu-

tants (Fig. 3G) identifies this receptor as a

major driver of mosquito attraction to warmth.

To test the specificity of theIr21amutant be-

havioral deficit for heat seeking, we examined

their ability to perform an activation-dependent

behavior less reliant on thermosensation. While

body heat is a powerful short-range cue, a multi-

modal combination of longer-range chemo-

sensory and visual cues mediates initial approach

( 3 ), suggesting that such behavior should be

largely unaffected by a specific thermosensory

Greppiet al.,Science 367 , 681–684 (2020) 7 February 2020 2of4

Fig. 1. IR21a is expressed in the antennal tip.
(A, upper)Ir21alocus, withIr21a+7bpmutations
shown in blue. (Lower) Virtual translations,
with 450 C-terminal amino acids of wild-type
IR21a replaced by 24 novel amino acids (blue)
and a premature stop inIr21a+7bp.(B) Targeted
integration generatingIr21aEYFP, a combined
bright-field and fluorescent illumination
image, and molecular genotyping results for
Ir21a+andIr21aEYFPhomozygotes. Lane m,
DNA size markers. (C) Mosquito anterior
[drawing based on ( 27 )]. Inset, flagellomere

13. (DandE) Immunostaining of flagellomere
    13 in wild-type (D) andIr21a+7bp(E) females.
    (n= 13 wild type;n=7Ir21a+7bp). Asterisks,
    IR21a-expressing cell bodies; arrows, sensory
    endings. HRP, horseradish peroxidase;
    DAPI, 4′,6-diamidino-2-phenylindole.
    Anti-HRP labels neuronal membrane
    proteins, and DAPI labels nuclei.

Fig. 2.Ir21ais required for thermosensing.
(A) Recording electrode insertion site. (B) Repre-
sentative recordings, with indicated regions
displayed on an expanded time scale. Circles,
spikes. The weighted average spike rate is the
instantaneous spike frequency smoothed
using a 1-s triangular window. Dotted lines, spike
thresholds. (C) Peri-stimulus time histograms
(averages ± SEM) for wild type (n=8)and
Ir21aEYFPandIr21a+7bp(n= 6) animals tested
at 30°C and 25°C. One heating-cooling trial
per animal. (D) Cooling response = (average
frequency 0.2 to 0.7 s after cooling onset)–
(average frequency 5 to 10 s precooling).
Heating response = (average frequency 0.5 to
1.5 s after heating onset)–(average frequency
5 to 10 s preheating). Lowercase letters
indicate distinct groups (Tukey’shonestsignifi-
cant difference,a= 0.01, except 32°C→37°C,
a= 0.05). Shapiro-Wilk test and analysis
of variance values are provided in the
statistics section of methods.

A Ir21aEYFP Ir21a+7bp

10 s

coolingwild typewarming

100 ms

25

31

60

0

weighted

ave. spike

rate (Hz)

Temp. ( ̊C)

1 mV

0

0.5

-0.5

mV

0

0.5

-0.5

mV

B

coeloconic

sensilla

C

10 s

a

b

37 ̊C 32 ̊C

Cooling response

32 ̊C 37 ̊C

Heating response

b

b

a a

b

b

Cooling response

30 ̊C 25 ̊C

Heating response

25 ̊C 30 ̊C

wild type

D

a

b

-20 02040 60

∆ spike rate (Hz)

-40-20 0

∆ spike rate (Hz)

b b

Ir21aEYFP

Ir21a+7bp

wild type Ir21aEYFP Ir21a+7bp

25 ̊C 30 ̊C

32 ̊C 37 ̊C

-20 02040 60

∆ spike rate (Hz)

-40-20 0

∆ spike rate (Hz)

wild type

Ir21aEYFP

Ir21a+7bp

60

0

40

20

80

100

spike

rate

(Hz)

20

30

Temp.^40

( ̊C)

20

Temp. 30

( ̊C)

40

60

0

40

20

80

100

spike

rate

(Hz)

37 ̊C 32 ̊C

30 ̊C 25 ̊C

20

20

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