deficit. To assess approach behavior, mosqui-
toes were activated by five human breaths
and presented a human hand, positioned on
a platform to prevent physical contact with
the mosquitoes but otherwise providing host-
associated cues (Fig. 4A). Hand approach
was strong in the wild type, with 57 ± 2% of
mosquitoes landing on the surface beneath
the hand (Fig. 4, B and C). Hand-associated
cues were critical, as hand withdrawal prompted
rapid dispersal (Fig. 4B).Ir21aEYFPmutants re-
mained robustly responsive, exhibiting max-
imum levels of approach (55 ± 2%) similar to
wild-type levels (Fig. 4, B and C). Careful ex-
amination of response kinetics revealed that,
compared to wild type, their initial accumula-
tion rate decreased by ~25% (Fig. 4, B and D),
and their dispersal rate upon hand removal
increased by ~40% (Fig. 4, B and E), poten-
tially reflecting subtle contributions of the
warmth gradient created by the presence of
the hand (fig. S4C) to the avidity of host ap-
proach. Similar results were obtained for
Ir21a+7bp(fig. S4, D to G). Overall, these data
demonstrate that the loss ofIr21adoes not
broadly disrupt orientation toward sensory
cues and argue against the presence of global
behavioral deficits in the mutants. These re-
sults are consistent with prior work indicating
that the disruption of single sensory modal-
ities is insufficient to completely eliminate host
approach ( 2 , 3 , 8 , 9 ).
Heat strongly stimulates mosquito blood
feeding ( 8 , 9 ). To assess the effect of warmth
on blood feeding, artificial membrane feeders
( 25 ) were used to present human blood mealsat different temperatures (Fig. 4F). One meal
was held at room temperature (RT, ~23°C)
andtheotherwarmedto~31°C,atempera-
ture similar to the surface temperatures (~29°C
to 33°C) of human torsos and extremities in
a23°Cto24°Croom( 26 ). In each trial, green
food coloring was added to one meal so that
the consumption of warm versus RT food
could be distinguished (fig. S5). Each class of
trial was assessed independently (RT meal
dyed green in Fig. 4G and fig. S4H; warm
meal dyed green in Fig. 4H and fig. S4I), and
each yielded similar results. In wild type, ele-
vated temperature robustly promoted feed-
ing, as reflected in the greater percentages of
mosquitoes consuming warm versus RT meals
(Fig. 4, G and H, and fig. S4, H and I). For
bothIr21aEYFP(Fig. 4, G and H) andIr21+7bpGreppiet al.,Science 367 , 681–684 (2020) 7 February 2020 3of4
Fig. 3.Ir21amediates heat seeking.(A) Heat
seeking assay. Assay box is 28 cm deep,
40 cm long, and 16 cm tall. (B) The stimulus
sequence and formula for the heat-seeking
index. (C) Representative images of 26°C (blue)
and 37°C (red) targets before and ~120 s
after CO 2 pulse initiation in wild type.
(DandE) Representative images from ~120 s
after CO 2 pulse initiation inIr21aEYFP(D)
andIr21a+7bp(E) mosquitoes. (F)Landing
on 37°C and 26°C targets as the percentage
of mosquitoes taking flight (averages ± SEM).
Wild type,n= 15 independent groups;Ir21aEYFP,
n=6;Ir21a+7bp,n= 7. There were 42 to
52 females per group. (G) Heat seeking
index (average from 105 to 135s). Letters
denote distinct categories (Steel-Dwass test,
P< 0.01). Shapiro-Wilk, Kruskal-Wallis, and
Steel-Dwass test values are provided in the
statistics section of methods.
Fig. 4.Ir21apromotes warmth-stimulated blood
feeding.(AtoE) Host approach. (A) Mosquitoes
were activated by five breaths and then presented a
hand. (B) Mosquitoes landed (accumulated) on the
cage roof below the hand. Values are averages ±
SEM for 33 to 75 females per assay. Wild type (wt),
n= 19 independent groups;Ir21aEYFP,n= 21. (C)
Average maximum accumulation, 180 s to 300s. (D)
Accumulation rate, {[(accumulation at 45 s)–
(accumulation pre–hand exposure)]/[(maximum
accumulation)–(accumulation pre–hand expo-
sure)]}/45 s. (E) Departure rate, 1–[(accumulation
at 340 s)/(accumulation at 300 s)]/40 s.
(FtoH) Blood feeding. (F) Two meals (one dyed
green) were placed on the cage. (G) RT meal
was dyed. (H) Warm meal was dyed. Dotted lines
link pairs. Total of 33 to 75 females per assay.
n= 6 independent groups per genotype, except in (H), whereIr21aEYFPn=7.Pvalues indicatettest results. The green tracking dye partially reduced relative
consumption of meals to which it was added. Cages are 17.5-cm-sided cubes. Shapiro-Wilk test values are provided in the statistics section of methods.
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