VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Behavioral Responses of Two Dengue Virus Vectors, Aedes aegypti and

Aedes albopictus (Diptera: Culicidae), to DUET and its Components
GARY G. CLARK,1,2 FRANCES V. GOLDEN,1 SANDRA A. ALLAN,1 MIRIAM F. COOPERBAND,3
4,5
AND JAMES R. MCNELLY

J. Med. Entomol. 50(5): 10591070 (2013); DOI: http://dx.doi.org/10.1603/ME12210

ABSTRACT Ultralow volume droplets of DUET, prallethrin, and sumithrin at a sublethal dose were
applied to unfed (nonbloodfed) and bloodfed female Aedes aegypti L. and Aedes albopictus (Skuse)
in a wind tunnel. Control spray droplets only contained inert ingredients. Individual mosquitoes were
videotaped before, during, and after spraying and various behaviors analyzed. During the spray periods
of all three pesticide treatments, mosquitoes spent a greater percentage of time moving, and the
distance moved was greater than for mosquitoes in the control treatments. In the postspray period,
the percent of time moving increased for mosquitoes exposed to all pesticide treatments compared
with the controls. After treatment, all females spent more time walking compared with controls, with
unfed Ae. aegypti females walking more after exposure to DUET and sumithrin than after exposure
to prallethrin and the control. Pesticide exposure increased ying in both species. Sumithrin exposure
increased activity and velocity of unfed mosquitoes more than bloodfed mosquitoes. DUET and
sumithrin treatments enhanced activity of Ae. aegypti females more than Ae. albopictus females.
KEY WORDS mosquito, Aedes aegypti, Aedes albopictus, DUET, prallethrin

Aedes (Stegomyia) aegypti L. is the principal epidemic

vector for dengue viruses in the world, with substantial
range expansion in the Americas during the last 40 yr
(Gubler 1998). Among other factors, this reinfestation
has contributed to the dramatic increase in dengue
and dengue hemorrhagic fever (DHF) in the western
hemisphere (San Martn et al. 2010). During the last
three decades, in total, 8,491,416 dengue cases, including 183,541 DHF cases, have been reported to the Pan
American Health Organization. The annual number of
cases caused by one or more of the four dengue serotypes in the Americas continues to rise annually. In
addition, Ae. aegypti can serve as the vector for yellow
fever (YF) virus in many of the Ae. aegypti-infested
urban centers of the Americas. In 19971998, after an
absence of 44 yr, the last urban YF outbreak was
reported from Santa Cruz, Bolivia (Van der Stuyft et
al. 1999). Although the YF virus infection was conrmed in only six residents, ve of these cases had a
fatal outcome, demonstrating the lethality of this viral
disease.
Although not considered as efcient a vector for
dengue viruses as Ae. aegypti, Aedes (Stegomyia) al1 Center for Medical, Veterinary, and Agricultural Entomology,
Agricultural Research Service, USDA, 1600 SW 23rd Dr., Gainesville,
FL 32608.
2 Corresponding author, e-mail: gary.clark@ars.usda.gov.
3 USDAAPHIS, PPQ, CPHST, 1398 W. Truck Rd., Buzzards Bay,
MA 02542.
4 Clarke, Roselle, IL 60172.
5 Current address: Volusia County Mosquito Control, 801 South St.,
New Smyrna Beach, FL 32168.

bopictus (Skuse) is also a competent vector of dengue

viruses (Lambrechts et al. 2010). Its importance as a
vector of dengue and YF viruses was reported after its
introduction into Brazil (Miller and Ballinger 1988)
and was exemplied by its ability to transmit 20
arboviruses in the laboratory (Paupy et al. 2009). The
presence of this second vector of YF virus in Brazil 10
yr after its discovery in the country has caused health
ofcials to be increasingly concerned about future
urban YF epidemics (Mondet et al. 1996). The role
and importance of Ae. albopictus as a vector of these
viruses, especially in laboratory studies of chikungunya virus (Turell et al. 1992), is further enhanced by
its dramatic expansion in geographic distribution in
the United States and internationally (Benedict et al.
2007).
One of the tools commonly used to control Ae.
aegypti females is the ultralow volume (ULV) application of insecticides (Reiter and Nathan 2001). Several studies of this vector species have reported that it
is frequently found indoors (Tidwell et al. 1990, Clark
et al. 1994, Kittayapong et al. 1997, Scott et al. 2000,
GarciaRejon et al. 2008) and beneath houses (Ponlawat and Harrington 2005) in urban areas, which can
be a factor in reducing the amount of ULV droplets
that penetrate the common resting sites (e.g., bedrooms, living rooms, and bathrooms) (Perich et al.
2000). Ae. albopictus has been found in urban, suburban, and rural areas (Lim et al. 1961) and has been
aspirated from vegetation around homes, bamboo
thickets, and areas directly adjacent to villages in Thailand (Ponlawat and Harrington 2005) where it is also

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frequently found indoors. Braks et al. (2003) reported

that in Brazil and Florida, Ae. aegypti was most prevalent in highly urbanized areas whereas Ae. albopictus
was found in rural, suburban, and vegetated urban
areas.
The application of a chemical that starts or speeds
up movement in these diverse adult resting sites, while
acting as a locomotor stimulant (Dethier et al. 1960),
may increase an excitatory behavioral response. This
term is reserved for activation of abnormally high
kinetic locomotion, like that produced by application
of sublethal amounts of certain insecticides in laboratory studies (Miller et al. 2009). In laboratory studies, Cooperband et al. (2010) exposed female Culex
quinquefasciatus Say to sublethal levels of prallethrin,
sumithrin, and piperonyl butoxide applied as ULV
droplets and found a positive correlation between
mosquitoes that spent more time ying during the
time of spray and number of insecticide droplets on
their bodies, presumably through increased contact
while ying. They also found that excitation in the
form of increased speed and duration of ight was
more immediate in mosquitoes exposed to prallethrin
than sumithrin. This behavior may improve the efcacy of peridomestic space spraying to control Ae.
aegypti and Ae. albopictus and impact dengue virus
transmission, an issue that was recently reviewed (Esu
et al. 2010).
The focus of this study was on three pyrethroid
insecticide treatmentsDUET (which contains prallethrin and sumithrin as active ingredients; Clarke,
Roselle, IL), prallethrin alone, and sumithrin alone.
These insecticides have been evaluated in eld studies
in the United States. Among other reports are those
with prallethrin (Responde) against three species of
mosquitoes in Arkansas and Louisiana (Groves et al.
1997) and with sumithrin (Anvil) against Anopheles
quadrimaculatus Say in Arkansas (Meisch et al. 2007).
More recently there has been the report of a eld
evaluation of DUET from Florida against West Nile
virus vectors, including Ae. albopictus (Qualls and Xue
2010).
The goal of this laboratory study was to determine
whether these insecticides, when applied as ULV
sprays, increased excitation in female Ae. aegypti and
Ae. albopictus that have or have not recently taken a
bloodmeal. Both species cause important public
health problems internationally and the use of ULV
insecticides that produce excitation and stimulate
them to move from their resting sites may have an
important role in their control.

Materials and Methods

Insects. Mosquitoes used for these studies were females from laboratory colonies of Ae. aegypti (Orlando
1952) and Ae. albopictus (Gainesville 2003) reared
(following Gerberg et al. 1994) and maintained at the
U.S. Department of Agriculture (USDA) Center for
Medical, Agricultural, and Veterinary Entomology in
Gainesville, FL.

Vol. 50, no. 5

Pupae were placed in cages (38.7 by 45.7 by 36.8

cm) (Bioquip, Inc., Rancho Dominguez, CA) maintained at 27C and 70% relative humidity (RH), and
with a photoperiod of 16:8 (L:D) h. Emerged adults
were provided 10% sucrose ad lib. Assays were conducted with 4- to 8-d-old females that were nonbloodfed (referred to as unfed) or recently bloodfed. Before
testing (30 150 min), unfed females were aspirated
from the emergence cage to a paper cylindrical carton
with a screened lid on the morning of the test. Bloodfed mosquitoes were fed 16 18 h before the bioassays. They were transferred by mouth aspirator to a
screened carton and were bloodfed for 1 h on debrinated bovine blood in a sausage casing (DeWeid,
San Antonio, TX) warmed by water bath to 40C and
placed on top of the carton. Females that had clearly
not bloodfed were discarded.
Wind Tunnel. This study was conducted in an
acrylic wind tunnel (30 by 30 by 120 cm) described in
detail in a previous study with Cx. quinquefasciatus
(Cooperband et al. 2010). This wind tunnel had a
detachable acrylic cylindrical spray chamber (15.6 cm
in diameter by 16.2 cm in height) that was used to
deliver pesticide spray into the wind tunnel from underneath with a plunger. An airbrush nozzle was inserted into the spray chamber through a small hole
(1.6 cm in diameter) mid-way up the side of the spray
chamber. After spraying into the chamber, the spray
hole was sealed with a piece of tape. ULV droplets
were produced by a single action airbrush (Badger,
model 350, Franklin Park, IL). To produce optimal
droplet sizes (8 30 m) as recommended on the
DUET label, a heavy tip and ne needle was used at
40 psi as determined previously by Cooperband et al.
(2010).
A small hole (1.3 cm in diameter) in the bottom of
the wind tunnel allowed spray from the spray chamber
to enter the wind tunnel. The hole was closed with an
acetate ap that was closed when the plunger was still.
As the plunger was pushed upward, spray from the
chamber moved into the wind tunnel. Each treatment
had its own spray chamber (and associated oor panel
of the wind tunnel) to ensure there was no crosscontamination. In addition, plastic wrap and aluminum foil used to cover the inside of the wind tunnel
was replaced between treatments.
A single mosquito was placed in a test chamber in
the wind tunnel. Air ow was measured with a hotwire
anemometer (TSI Incorporated, Shoreview, MN) inside and to the side of the test chamber. Beside the test
cage, the wind speed was 100 cm/s, while the mesh
reduced the wind speed to 50 cm/s inside the test
chamber, a speed at which droplet movement is enhanced (Hoffmann et al. 2008). Experiments were
conducted at 22C and 60% RH.
Test Chambers. Test chambers to contain mosquitoes within the wind tunnel during their exposure to
treatments were fabricated from 0.95-liter paper cylinders (Cooperband et al. 2010). Briey, both ends
and one side of the carton were removed and replaced
with nylon mesh to allow airow and pesticide delivery. A clear acetate sheet was glued to the open side

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Table 1. Constituents of the four treatments used for evaluation against female Ae. aegypti and Ae. albopictus in wind tunnel
assays
Treatment
DUETa
Prallethrin
Sumithrin
Control

Components (%)
Prallethrin

Sumithrin

PBO

Inert

1
1

5
5
5

89
94
90
100

PBO is piperonyl butoxide; inert consists of a proprietary mixture

of inert ingredients;indicates absence of an ingredient.
a
Full formulation of DUET.

so that the behavior of the mosquito could be videotaped. The bottom of the test chamber had a hole (1
cm in diameter) corresponding to a similar hole in the
bottom of the wind tunnel for introduction and removal of individual mosquitoes.
Bioassay Procedures. Each treatment was conducted with a single (4- to 8-d-old) female mosquito
(Ae. aegypti or Ae. albopictus) that was either unfed or
bloodfed. The test chamber containing the single female mosquito was placed 81.3 cm downwind of the
spray chamber in the wind tunnel and located in the
air stream from the spray chamber so that droplets
from the spray chamber moved through the carton.
The optimal location of the test chamber within the
wind tunnel was determined previously using sublimated dry ice vapor. Conditions for experimentation
were described previously by Cooperband et al.
(2010). After the test chamber was placed in the wind
tunnel, the mosquito was allowed to come to a resting
position within the test chamber before the test and
the prespray period began.
Responses of the mosquitoes within the test chamber to the treatments were video-taped for subsequent
analysis using a CCD video camera (Panasonic, model
WV-BP334, Secaucus, NJ) with an automatic iris lens
(CCTV, one-third inch, 3 8 mm, F 1.4, Rainbow, Irvine, CA), an MPEG recorder (Canopus Co., model
EMR100, Kobe, Japan), and laptop computer. The
video recording for each female lasted 7.5 min and
consisted of three periods: prespray, during spray, and
postspray. The prespray period recorded mosquito
movement for the 2-min period before the initiation of
spraying. The start and end of the spray period was
perceptible on the video recording. The spray period
consisted of depressing the airbrush for 0.5 s to load
the spray chamber with droplets, and then the 2.5 min
to plunge the droplets into the wind tunnel. The following 3 min consisted of the postspray period for
observation of mosquito movement in the absence of
droplets.
Treatments. Sprays consisted of four treatments,
including the full DUET formulation and three subformulations, which consisted of various combinations
of ingredients, as supplied by Clarke Mosquito Control
(Roselle, IL) (Table 1). Treatments were made at a
sublethal exposure level of the formulations at a 0.5 s
spray. Doses were measured in a settling chamber, and
doses were selected that did not cause immediate

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knockdown but resulted in 50% mortality at 24 h

(Cooperband et al. 2010). The air in the spray chamber was cleared for 510 min between replicates of
similar treatments. In addition, the foil and plastic
linings of the wind tunnel were removed and replaced,
and the airbrush was thoroughly cleaned with acetone. Lack of residual effects from the previous treatment was veried by minimal amount of mosquito
movement observed in the prespray portion of each
video. Ten replicates of each treatment were performed, with a total of 160 females being treated and
recorded (10 females 2 species 2 bloodfed
states 4 treatments).
Assessment of Knockdown. After treatment, each
mosquito was removed from the test chamber and
placed in a new screened paper carton for observation
where knockdown (KD) was assessed every 30 min
over an 8-h period. A mosquito was considered
knocked down if it was unable to stand and was not
differentiated from death.
Analysis of Video Recordings. Videotapes were edited into three periods (prespray, during spray, and
postspray), contrast enhanced, and reduced in size to
ve frames per s (Power Director [version 6], Cyberlink, Taipei, Taiwan). Video recordings were analyzed
for movement analysis (Motus [version 8.2], Peak
Performance Technologies, Inc., Centennial, CO, and
Ethovision [version 7.0], Noldus Information Technology, Wageningen, The Netherlands) and specic
behaviors (The Observer XT [version 8.0], Noldus
Information Technology).
The movement track of each mosquito was manually digitized to produce a movement track from
which the two-dimensional vectors, velocity, and percent of time spent in motion could be calculated. From
this initial analysis, the percent of movement (based
on number of frames in which movement occurred
from the previous frame starting with the initial
frame) and the average velocity while moving (including ying and walking) was determined. From
this, velocity 1 cm/s, previously determined to be an
effective assessment of pesticide impact on movement
(Cooperband et al. 2010), was calculated. Movement
tracks from Motus were analyzed in Ethovision to
measure the distance moved.
Using behavioral analysis software (The Observer
XT), the time that a mosquito spent in motion (walking and ying), or not in motion (resting) was determined for the three treatment periods and converted
to percent time for each treatment period to provide
the percentage of time walking, ying, resting, and
knocked down.
Statistical Analyses. Data were analyzed within
each of the two spray periods (during and after spray)
using an analysis of variance (ANOVA) model to identify differences between the xed effects of mosquito
species, physiological state (unfed, bloodfed), and
treatment (formulation of DUET and its components), and interactions. Nonsignicant interactions
were dropped from the model and data reanalyzed
(ANOVA PROC GLM) (version 9.2, SAS Institute,
Cary, NC). For parameters with no signicant effects

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Fig. 1. (A) Percentage of mosquitoes that were knocked-down (KD) or dead in each treatment over time. Ten
mosquitoes were subjected to each treatment. A decrease in the percentage indicates recovery from KD. (B) Percent of time
spent knocked-down (KD) (SE) by female Ae. aegypti and Ae. albopictus in the postspray period. Treatment bars with
different letters indicate signicant differences (P 0.05).

or interaction with state, data from unfed and bloodfed mosquitoes were combined for gures and subsequent analyses. Before analysis, data were analyzed
with Levenes test for homogeneity of variance and
data log (x1) or arcsine transformed as necessary
with untransformed means presented in tables and gures. Post hoc means comparison tests were performed
using the StudentNeumanKeuls test or paired t-tests
using a Bonferroni adjustment to (P 0.05). Within
each treatment, means for unfed and bloodfed females
were compared by paired t-test (P 0.05).

Results
The KD and mortality responses (Fig. 1A) produced in unfed and bloodfed Ae. aegypti following
exposure to DUET at the selected sublethal dose were
quite similar. The responses of unfed and bloodfed Ae.
albopictus were also similar, yet different from those of
Ae. aegypti. The KD and mortality responses following
exposure to prallethrin were similar in the bloodfed
Ae. aegypti and Ae. albopictus. A similar response pattern was observed in the unfed Ae. aegypti and Ae.

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Fig. 2. Effect of pesticide treatments in a wind tunnel on percent movement (SE) of female Ae. aegypti and Ae. albopictus
in the pre-, during-, and postspray periods. If no differences were detected between responses of unfed and bloodfed, means
were combined. Treatment bars of the same color with different letters indicate signicant differences (P 0.05). Asterisks
indicate signicant differences between unfed and bloodfed mosquitoes for a particular treatment (paired t-test; P 0.05).

albopictus. The KD and mortality response following

exposure to sumithrin was similar in the bloodfed Ae.
aegypti and Ae. albopictus. A similar response pattern
was observed in the unfed Ae. aegypti and Ae. albopictus exposed to sumithrin. The overall control mortality
for all sprays was 12%. There was little KD (3 out of 40
individuals) during the spray period, with no differences for knockdown between species, states, or treatments (P 0.05) (Fig. 1B). In the postspray period,
however, only treatment signicantly affected responses (F3, 154 12.49; P 0.01). Knockdown of Ae.
aegypti females was similar after exposure to DUET
and prallethrin and greater than to sumithrin, whereas
knockdown after exposure to prallethrin was greater
than other treatments for Ae. albopictus (Fig. 1B).
The effect of state (unfed vs. bloodfed) was generally not signicant in any of the main effect comparisons; however, several two-way interactions with

state were signicant. For parameters that had no

signicant effects involving state, gures are presented with data for unfed and bloodfed females combined. For parameters with signicant differences involving state, data for unfed and bloodfed females are
presented separately.
Percent movement, determined as the percent of
video frames indicating movement, provided insight
into the effects of the pesticide treatments. There was
little mosquito movement in the prespray period, with
no signicant differences among the control and pesticide treatments (P 0.05), and prespray data were
excluded from further analysis (Fig. 2). During the
spray period, however, model effects for treatment
(F3, 148 23.93; P 0.001) and species (F1, 148 25.44;
P 0.001) were signicant. However, signicant interactions between treatment species (F3, 148 3.53;
P 0.05) and treatment state (F3, 148 4.32; P

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Fig. 3. Effect of pesticide treatments in a wind tunnel on moving velocity 1 cm/s (SE) of Ae. aegypti and Ae. albopictus
females in the during and postspray periods. If no differences were detected between responses of unfed and bloodfed, means
were combined. Treatment bars of the same color with different letters indicate signicant differences (P 0.05). Asterisks
indicate signicant differences between unfed and bloodfed mosquitoes for a particular treatment (paired t-test; P 0.05).

0.01) indicate that treatment effects vary with species

and state. Because of the signicant state interaction
during the spray period, means for unfed and bloodfed
mosquitoes are presented separately (Fig. 2). All pesticide treatments resulted in higher percent movement of both unfed and bloodfed Ae. aegypti compared with controls (Fig. 2). For Ae. albopictus, all
pesticide treatments enhanced movement, whereas
only prallethrin treatments increased movement of
bloodfed mosquitoes. Exposure to DUET resulted in
more movement by unfed and bloodfed Ae. aegypti
compared with Ae. albopictus (P 0.05). Exposure to
sumithrin resulted in more movement in unfed Ae.
albopictus females compared with bloodfed females
(t18 3.72; P 0.01). During the postspray period,
model effects for treatment (F3, 151 42.62; P 0.001)
and species (F1, 151 26.52; P 0.001) were signicant, with signicant species treatment interactions
(F3, 151 7.11; P 0.001). There were no differences
between unfed and bloodfed mosquitoes, and these
data were pooled. For Ae. aegypti, sumithrin spray in
the postspray period resulted in more movement than
DUET and prallethrin sprays, which, in turn, resulted
in more activity than the controls (Fig. 2). In contrast,
female Ae. albopictus moved to the same extent after
exposure to all pesticide sprays and more than the
controls (Fig. 2). Overall, female Ae. aegypti moved
more than Ae. albopictus after exposure to both DUET
(t18 4.78; P 0.001) and sumithrin (t18 4.84; P
0.001) sprays.
Velocity of movement (1 cm/s) during the spray
period had signicant main effects for treatment

(F3, 151 19.18; P 0.001), with signicant interactions of species and treatment (F3, 151 4.85; P 0.01)
(Fig. 3). All pesticide treatments increased moving
velocity compared with the control during the spray
period for both mosquito species (Fig. 3). Velocity of
Ae. aegypti was greater than Ae. albopictus after exposure to DUET (t18 3.42; P 0.05). After the spray
period, main effects for treatment (F3, 148 45.67; P
0.001) and species (F1, 148 13.43; P 0.001) were
signicant; however, there were also signicant interactions between species and treatment (F3, 148 6.28;
P 0.001) and state and treatment (F3, 148 3.17; P
0.05). After spraying with pesticides, Ae. aegypti females moved more quickly than control mosquitoes
(Fig. 3). All insecticide treatments increased moving
velocity of unfed Ae. albopictus, whereas only bloodfed females exposed to prallethrin increased velocity.
Bloodfed Ae. aegypti females increased velocity more
than bloodfed Ae. albopictus females after exposure to
DUET spray (t18 3.94; P 0.001). Velocity increased
more in unfed female Ae. albopictus compared with
bloodfed females with exposure to sumithrin (t18
3.29; P 0.05).
The distance traveled by mosquitoes during the
spray period had signicant main effects of pesticide
treatment (F3, 151 32.66; P 0.001) and mosquito
species (F1, 151 20.46; P 0.001), with signicant
interactions of species treatment (F3, 151 9.34; P
0.001). During the spray period, exposure to all of the
pesticide treatments resulted in more distance covered by Ae. aegypti compared with controls (Fig. 4).
For Ae. albopictus females, responses were greatest to

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Fig. 4. Effect of pesticide treatments in a wind tunnel on distance covered (centimeter) (SE) by female Ae. aegypti
and Ae. albopictus in the during and postspray periods. Treatment with different letters indicate signicant differences
(P 0.05).

prallethrin sprays (Fig. 4). Female Ae. aegypti traveled

a greater distance than Ae. albopictus females in the
presence of DUET (t18 4.15; P 0.01) and sumithrin
(t18 3.48; P 0.05). In the postspray period, the
main effects of treatment (F3, 151 33.59; P 0.001)
and species (F1, 151 15.22; P 0.001) were signicant
with signicant interaction between species and treatment (F3, 151 5.71; P 0.01). Female Ae. aegypti
covered more distance after exposure to sumithrin
compared with DUET and prallethrin, both of which
resulted in more distance covered than the control
(Fig. 4). For Ae. albopictus, all pesticide treatments
resulted in greater distances traveled than the controls
(Fig. 4). Females of Ae. aegypti moved greater distances than Ae. albopictus females after exposure to
sprays containing DUET (t18 4.71; P 0.001) or
sumithrin (t18 5.03; P 0.001).
The percent of time spent resting during the spray
periods had signicant main effects of treatment
(F3, 148 20.49; P 0.001) and mosquito species
(F1, 148 20.09; P 0.001), with signicant interaction
between species and treatment (F3, 148 3.45; P
0.05) and state and treatment (F3, 148 3.37; P 0.05)
(Fig. 5). For Ae. aegypti, all pesticide treatments resulted in less resting than controls (Fig. 5). For Ae.
albopictus, resting was signicantly reduced after exposure to prallethrin (unfed and bloodfed females)
and sumithrin (unfed females) compared with controls (Fig. 5). Unfed females of Ae. albopictus rested
more than Ae. aegypti females during exposure to
DUET (t18 3.92; P 0.05) and sumithrin (t18 3.72;
P 0.05). Resting was lower for unfed Ae. aegypti

females compared with bloodfed females during

sprays with sumithrin (t18 3.36; P 0.001). After the
spray period, main effects of treatment (F3, 151 42.29;
P 0.01) and species (F1, 151 45.64; P 0.001) and
species by treatment interactions (F3, 151 5.63; P
0.001) were detected (Fig. 6). Resting by Ae. aegypti
females, reduced by about one-half in all pesticide
treatments, was lower than the untreated controls
(Fig. 6). Although resting of Ae. albopictus females
after exposed to pesticides was lower than the controls, there was only a 20 30% reduction. Resting of
Ae. aegypti females was more reduced than for Ae.
albopictus females in the presence of DUET (t18
6.17; P 0.001) and sumithrin (t18 4.48; P 0.05)
sprays.
The percent of time spent walking during the spray
period had signicant main effects of treatment
(F3, 152 19.86; P 0.001), species (F1, 152 19.48; P
0.001), and interactions between treatment and state
(F3, 152 3.35; P 0.05) (Fig. 5). For unfed and
bloodfed Ae. aegypti and unfed Ae. albopictus, more
time was spent walking during the pesticide sprays
compared with the control sprays (Fig. 5). For bloodfed Ae. albopictus, this increase in walking was only
seen with DUET and prallethrin treatments. Bloodfed
Ae. aegypti spent more time walking than did bloodfed
Ae. albopictus during the DUET sprays (t18 5.46; P
0.05). Also, unfed Ae. aegypti spent more time walking
than did unfed Ae. albopictus during sumithrin sprays
(t18 5.19; P 0.05). Unfed Ae. aegypti females spent
more time walking while being sprayed with sumithrin
compared with bloodfed mosquitoes (t18 4.07; P

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Fig. 5. Effect of pesticide treatments in a wind tunnel on percentage of time during the spray period that female Ae. aegypti
and Ae. albopictus spent resting, walking, or ying. If no differences were detected between responses of unfed and bloodfed,
means were combined. Treatment bars of the same color with different letters indicate signicant differences (P 0.05).
Asterisks indicate signicant differences between unfed and bloodfed mosquitoes for a particular treatment (paired t-test;
P 0.05).

0.01). In the postspray period, the percent of time

walking had signicant main effects of pesticide treatment (F3, 154 38.61; P 0.001) and species (F1, 154
50.18; P 0.001) (Fig. 6). All pesticide treatments
resulted in more walking by female Ae. aegypti exposed to DUET and sumithrin compared with those
treated with prallethrin, which walked more than the
controls (Fig. 6). For Ae. albopictus, all pesticide treatments resulted in more time walking than the controls.
Female Ae. aegypti responded to all pesticide treatments, with signicantly more walking than Ae. albopictus females (P 0.05).

The percent of time in ight during the spray period

had signicant main effects of treatment (F3, 151
10.76; P 0.001) and species (F1, 151 5.72; P 0.05),
with signicant interaction of species and treatment
(F3, 151 6.23; P 0.001) (Fig. 5). All pesticide treatments increased the percent of time ying by Ae.
aegypti, in contrast to prallethrin, which was the only
pesticide increasing ight of Ae. albopictus. Greater
increases in ight occurred with Ae. aegypti compared
with Ae. albopictus for DUET (t18 3.54; P 0.001)
and sumithrin (t18 3.11; P 0.01) treatments. In the
postspray period, the percent of time in ight had

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CLARK ET AL.: BEHAVIORAL RESPONSE OF Aedes to DUET

1067

Fig. 6. Effect of pesticide treatments in a wind tunnel on percentage of time in the postspray period that female Ae. aegypti
and Ae. albopictus spent resting, walking, or ying. If no differences were detected between responses of unfed and bloodfed,
means were combined. Treatment bars of the same color with different letters indicate signicant differences (P 0.05).
Asterisks indicate signicant differences between unfed and bloodfed mosquitoes for a particular treatment (paired t-test;
P 0.05).

signicant main effects of treatment (F3, 148 16.52;

P 0.001), species (F1, 148 8.71; P 0.01), and state
(F1, 148 12.13; P 0.001), with signicant interactions of species and treatment (F3, 148 8.56; P
0.001) and state and treatment (F3, 148 2.81; P 0.05)
(Fig. 6). The percent of time ying by unfed Ae.
aegypti was twofold greater after sprays containing
sumithrin than to other pesticide treatments (Fig. 6)
and exposure to all pesticides increased ight compared with controls. For Ae. albopictus, however, ight
was enhanced modestly in response to pesticide exposure (Fig. 6). In addition, after exposure to sumi-

thrin, ight was increased more for unfed female Ae.

aegypti (t18 4.03; P 0.01) and Ae. albopictus (t18
3.50; P 0.01) females than for bloodfed females.
Discussion
Populations of mosquitoes consist of individuals in
a variety of different ages and physiological states (i.e.,
teneral, unfed, bloodfed, and gravid), with differences
in insecticide susceptibility reported among different
ages (Davidson 1958, Rowland and Hemingway 1987,
Hodjati and Curtis 1999, Glunt et al. 2011, Rajatileka

1068

JOURNAL OF MEDICAL ENTOMOLOGY

et al. 2011), between sexes (Davidson 1958, Doyle et

al. 2009), and among blood-feeding status (Davidson
1958, Rawlings et al. 1981, Polsomboon et al. 2008).
Within homes or their immediate surroundings, the
common physiological states of Ae. aegypti and Ae.
albopictus are unfed and freshly bloodfed individuals
(Scott et al. 2000). Previous studies have indicated that
insecticide susceptibility is lowest just after bloodfeeding (24 h for Ae. aegypti, 48 h for Culex, and 72 h
for Culiseta: Eliason et al. 1990), with a gradual increase in susceptibility in unfed females (Eliason et al.
1990, Rajatileka et al. 2011). Hadaway and Barlow
(1956) reported similar results for Ae. aegypti and
Anopheles stephensi Liston to DDT. Ramications of
this have been reported previously (cited in Eliason et
al. 1990) with the rapid appearance of egg rafts and
larvae shortly after a spray application, indicating that
the application did not effectively kill bloodfed and
parous adults. Because of the difculty in effectively
controlling these recently bloodfed females, it was
important to compare the sublethal exposure of unfed
and bloodfed Ae. aegypti and Ae. albopictus females to
the individual and combined pyrethroids in this study
on ight and thus the potential exposure to ULV droplets during the period of time before the insecticide
spray has settled (Cooperband et al. 2010).
Overall, for nearly all of the parameters measured,
the unfed or bloodfed state was not signicant in
analyses, indicating that all pesticide spray treatments
were as effective against unfed females as against
bloodfed females. The exception was the higher ight
activity observed with unfed females compared with
bloodfed females. Unlike the other pesticide treatments and indicated by signicant state and treatment
interactions, sumithrin exposure enhanced activity of
unfed females with increased time walking and ying
during spray periods, greater velocity of movement,
more ight, and less time resting after the spray period.
Sprays of prallethrin and DUET were equally effective
in activating bloodfed and unfed females of both species.
The species of target mosquitoes can clearly affect
the efcacy of treatment as demonstrated in laboratory (Pridgeon et al. 2008, Cooperband and Allan
2009) and eld studies (Groves et al. 1997, Trout et al.
2007). Behavioral studies on sublethal exposure to
different pyrethroids demonstrated differences in
ight duration, velocity, turn angle, and angular velocity in three species of mosquitoes (Cohnstaedt and
Allan 2011). In the current study with sublethal exposures to these pesticides, species was a signicant
factor in various behavioral attributes (e.g., percent of
time in movement, time resting, and distance moved),
with Ae. aegypti more responsive and active in the
presence of DUET and sumithrin sprays than Ae. albopictus. However, there was no difference between
species in the percent of time knocked down. Clearly,
species can affect some aspects of behavioral response
to pesticides, particularly at sublethal doses. As indicated by signicant interaction effects, the behavioral
responses to pesticide treatments differed between
species. Female Ae. aegypti in the presence of DUET

Vol. 50, no. 5

or sumithrin sprays demonstrated more activity (percent movement, moving velocity, distance covered,
and percent of time ying and walking) in some of the
measured parameters than Ae. albopictus females.
Conversely, Ae. albopictus spent more time resting
after those treatments.
Similar to Cooperband et al. (2010), results in this
study support the role of the two pyrethroid insecticides (prallethrin and sumithrin) as locomotor stimulants (Dethier et al. 1960). The activating effect of
sumithrin in the postspray period documented in
Culex (Cooperband et al. 2010) was evident in unfed
Ae. aegypti but not in bloodfed or unfed Ae. albopictus.
In a prior laboratory study evaluating DUET and its
components on behavior of Cx. quinquefasciatus, excitation in the form of increased speed and ight
duration was immediate in mosquitoes exposed to
prallethrin (Cooperband et al. 2010). In contrast to the
other treatments in that study containing prallethrin
or DUET, exposure to sumithrin did not increase exposure to ULV droplets. As mosquito species vary with
their responsiveness to sublethal exposures of pyrethroids, with Culex tending to respond or act more
slowly than other species after exposure (Cooperband
and Allan 2009, Cohnstaedt and Allan 2011), it was
unclear how Aedes mosquitoes would respond to
DUET and its components. Similar to previous studies
with unfed Culex (Cooperband et al. 2010), unfed Ae.
aegypti and Ae. albopictus females exposed to prallethrin and DUET moved more than controls both
during and after spray periods. In a bottle-assay evaluation on the contact toxicity of DUET using eldcollected mosquitoes, Qualls and Xue (2010) reported
differences in species susceptibility; Ae. albopictus,
Aedes taeniorhynchus (Wiedemann), and Psorophora
columbiae (Dyar & Knab) were more susceptible than
Cx. quinquefasciatus.
The value of ULV insecticide treatments that increase the movement and ight of exposed mosquitoes
is clear particularly with respect to control efforts that
target mosquitoes in unexposed and semiexposed
places (Perich et al. 2000, Khadri et al. 2009). Peridomestic spraying remains a widely used tool for mediation of dengue transmission (Esu et al. 2010), and
enhancement of efcacy of control of bloodfed as well
as unfed females could greatly improve population
reductions. Enhanced mosquito ight can result in
greater exposure to ULV droplets (Cooperband et al.
2010). The behavioral (excitation) responses of Ae.
aegypti and Ae. albopictus to excitatory active ingredients such as prallethrin and sumithrin expose them
to more ULV droplets and reects their potential value
in controlling these dengue virus vectors. This impact
might best be seen when they were used in outdoor
habitats with Ae. albopictus present such as in Singapore (Lim et al. 1961), Thailand (Ponlawat and Harrington 2005), and Hawaii (Efer et al. 2005). Furthermore, Ae. aegypti control might be enhanced
through the indoor use of these pyrethrins (if approved by appropriate regulatory agencies) where
others have been used previously in India (Mani
et al. 2005), Taiwan (Teng et al. 2007), Thailand (Pant

September 2013

CLARK ET AL.: BEHAVIORAL RESPONSE OF Aedes to DUET

et al. 1974, Koenraadt et al. 2007), Honduras (Perich

et al. 2001), and Costa Rica (Perich et al. 2003). Regardless, it should be apparent that a single approach
to public health vector control will not eliminate these
important vectors of human and animal pathogens but
should be considered as part of an integrated vector
management framework (WHO 2004).
Acknowledgments
We thank Bill Jany for stimulating discussions, Erin Vrzal
Wessels for assisting with the experimental analysis, Haze
Brown for providing the mosquitoes, and manuscript review
by Dan Kline and Ruide Xue. This study was conducted
under a nonfunded cooperative agreement with Clarke Mosquito Control, Inc.

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Received 21 September 2012; accepted 11 July 2013.