Dengue Infection Increases the Locomotor Activity of
Aedes aegypti Females
Tamara N. Lima-Camara1., Rafaela V. Bruno1., Paula M. Luz2, Ma ´rcia G. Castro3, Ricardo
Lourenc ¸o-de-Oliveira3, Marcos H. F. Sorgine4,5, Alexandre A. Peixoto1,5*
1Laborato ´rio de Biologia Molecular de Insetos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, 2Laborato ´rio de Pesquisa Clı ´nica em DST & AIDS, Instituto de
Pesquisa Clı ´nica Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brazil, 3Laborato ´rio de Transmissores de Hematozoa ´rios, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro,
Brazil, 4Programa de Biologia Molecular e Biotecnologia, Instituto de Bioquı ´mica Me ´dica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 5Instituto
Nacional de Cie ˆncia e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Brazil
Background: Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in
importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other
parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior
of Aedes aegypti females with respect to their activity level.
Methods/Principal Findings: We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and
recorded their locomotor activity behavior. We observed an increase of up to ,50% in the activity of infected mosquitoes
compared to the uninfected controls.
Conclusions: Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity
observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether
this behavioral change could have implications for the dynamics of Dengue virus transmission.
Citation: Lima-Camara TN, Bruno RV, Luz PM, Castro MG, Lourenc ¸o-de-Oliveira R, et al. (2011) Dengue Infection Increases the Locomotor Activity of Aedes aegypti
Females. PLoS ONE 6(3): e17690. doi:10.1371/journal.pone.0017690
Editor: Laurent Re ´nia, Agency for Science, Technology and Research - Singapore Immunology Network, Singapore
Received December 3, 2010; Accepted February 10, 2011; Published March 8, 2011
Copyright: ? 2011 Lima-Camara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Howard Hughes Medical Institute, CNPq, Faperj, and FIOCRUZ. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Over the last decades, Dengue outbreaks have been a major
public health concern in many parts of the World, where Dengue
epidemics have been registered with a significant number of deaths
. There are four antigenically distinct RNA viruses that can
cause the disease, and in Brazil, three Dengue serotypes (DENV-1;
DENV-2 and DENV-3) have co-circulated in several areas and
caused some severe Dengue epidemics .
Aedes aegypti (Diptera: Culicidae) is the urban vector of Yellow
Fever and Dengue viruses. This diurnal mosquito is very
anthropophilic and endophilic, being commonly found in urban
and suburban areas, especially where house and human densities
are high and where it seems to live longer and disperse to short
distances (e.g. –).
It is known that parasites can alter vector behavior (reviewed by
–) and a number of studies have reported behavioral changes
in Ae. aegypti infected with pathogens and symbionts. For example,
Rossignol et al  observed that Ae. aegypti females experimentally
infected with an avian malaria parasite, Plasmodium gallinaceum, take
more time to locate blood in guinea pigs than the uninfected ones.
Rowland and Lindsay  studied the flight activity of females of
this species infected with the filarial parasite Brugia pahangi and
observed a decrease in flight capacity in heavily-infected
mosquitoes under laboratory conditions. Recently, it has been
shown that the infection by the symbiotic bacterium Wolbachia can
also drastically alter this mosquito’s behavior and physiology –
In the current study, we investigated whether infection with
Dengue virus causes changes in the locomotor activity behavior of
Ae. aegypti females under laboratory conditions.
Infection of Mosquitoes with the Dengue virus
The Paea strain of Ae. aegypti was used in all infection
experiments. This laboratory strain is known to be highly
susceptible to Dengue virus serotype 2 (DENV-2) infection .
Mosquito colony was reared according to procedures described in
. Males and females remained together and were fed with 15%
sucrose solution since emergence.
Five-day-old female mosquitoes were individually infected by
intrathoracic inoculation with 0.21 ml of L-15 (Leibovitz)
Medium containing Dengue virus (DENV-2 strain FIOCRUZ-
66985 ) in a concentration of 107PFU using a Nanoject
microinjector (Drummond Scientific). Control mosquitoes were
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intrathoracically inoculated with 0.21 ml of only L-15 (Leibovitz)
To verify the success of the experimental infections, the heads of
a number of mosquitoes that were inoculated with virus and that
were alive by the end of the locomotor activity experiments
(around 8–10 days after inoculation), plus negative controls, were
tested for Dengue infection via RT-PCR, as described below. The
results indicated that over 95% (70/73) of the mosquitoes
inoculated with the Dengue virus were infected (data not shown).
Detection of Dengue virus in mosquitoes
Mosquito heads were separated from bodies on a metal plate
placed on dry ice and viral RNA was extracted from each head
using the QIAmp Viral Mini Kit (Qiagen) according to the
manufacturer’s protocol. RT-PCR for detecting DENV2 was
conducted in a PCR System 9700 GeneAmp (Applied Biosystems)
using SuperScriptTMOne-Step RT-PCR with PlatinumH Taq
(Invitrogen) and Dengue virus consensus primers D1 and D2 ,
followed by a semi-nested PCR on the resulting product using Go
Taq Green Master Mix (Promega) and primers D1 and TS2 .
PCR products were electrophoresed on 2.5% agarose gels. A band
of 119 pb corresponding to DENV-2 could be seen under UV
light in the infected mosquitoes.
Analysis of the locomotor activity behavior
The activity of infected and uninfected control Ae. aegypti females
was recorded using a larger version of the Drosophila Activity
Monitor (TriKinetics) as described in . After inoculation with
Dengue virus or L-15 medium, females were individually placed in
glass tubes (1 cm67 cm) with a cotton plug soaked in 15% sucrose
solution and these tubes placed in the Activity Monitor inside a
Precision Scientific Incubator Model 818 under a constant
temperature of 25uC and a photoperiod of 12 hours of light and
12 hours of dark (LD 12:12). For each mosquito, the total
locomotor activity of 1 hour-intervals was recorded for about
seven days after inoculation. The data of individuals that died
during the experiments were excluded, and the data analysis was
carried out comparing the activity data of infected and uninfected
mosquitoes from the second to sixth day after inoculation.
Table 1 shows the mean hourly locomotor activity of control
and Dengue virus infected females in four different experiments.
We observed that infected females of Ae. aegypti showed more
activity than controls in all experiments. The relative increase in
activity ranged from ,10% to more than 50%. A two-way
ANOVA indicated a significant difference in the activity between
infected and uninfected control females (p,0.01). Although a
significant difference in the overall activity levels was also observed
between experiments (p,0.01), the interaction between experi-
ments and infection was not significant (p=0.82) indicating that
the difference between infected and uninfected females was
Figure 1 shows the normalized locomotor activity patterns of
infected and control females during a full LD 12:12 cycle (Fig. 1A)
or during the photophase (Fig. 1B). As previously reported in the
literature (reviewed in ), Ae. aegypti is a diurnal species showing
higher activity levels towards the end of the photophase (‘‘late
afternoon’’) and a characteristic startle response to the abrupt
light-on/light-off transition . The comparison of the normal-
ized locomotor activity patterns of infected and uninfected females
shows that Dengue infection causes an increase in activity
throughout the 24 hour period. Although this effect is most
dramatic during the light-on/light-off transition (Fig. 1A), an
increase in activity is also seen throughout the day and night in
infected females, especially during the ‘‘natural’’ activity peak
occurring around ZT 9 and in the last hours of the photophase
(Fig. 1B). In fact, this increase in activity associated with Dengue
infection is still significant (p=0.012) even when we exclude the
light-on/light-off transition (Table 1). In summary, our results
indicate that the locomotor activity of infected females is
consistently increased when compared to that of uninfected
Very little is known about the effects of viral infection on Aedes
mosquitoes. Several authors have shown that Dengue virus exhibits
Table 1. Activity increase in Dengue infected Aedes aegypti females.
ExperimentStatusN Mean activity per hour* (± SEM)
Relative increase of activity in
infected mosquitoes (%)
1 Control17 5.0661.16
2 Control 53 11.2261.45
3 Control 459.8461.19
4 Control 7011.8861.01
Infected 83 13.4661.13
*The numbers in parenthesis refer to the activity excluding the light-on/light-off transition.
Dengue and Mosquito Activity
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a remarkable tropism for the mosquito nervous tissues. Linthicumet
al  studied the tropism of DENV-3 in parenterally infected
female Aedes aegypti mosquitoes using immunocytochemical methods
and observed that the nervous tissues were among the first tissues to
be infected. In fact, these authors suggested that the nervous system
is the primarysite ofvirusamplification inmosquitoes infected using
thismethod . Several years later,Salazar et al  corroborated
these findings by showing that in mosquitoes orally infected with
DENV-2, the nervous tissues are among the first to be infected,
presenting detectable levels of viral antigens 5 days after an infective
blood meal. Interestingly, these authors also showed that heads and
salivary glands were the only tissues where viral antigens continued
to accumulate throughout the 21 days observed in their study. All
other mosquito infected tissues presented a decrease in the infection
This remarkable tropism of Dengue virus for the insect nervous
tissues led us to hypothesize that the infection might have some
role in modulating the vector locomotor activity behavior, since it
is known that activity rhythms in Drosophila and other Diptera are
regulated by circadian clock neurons in the brain (reviewed in
,). In fact, our results show that although the daily activity
patterns of DENV-2 infected and uninfected mosquitoes are
similar, the total level of activity is clearly increased upon infection.
This increase is most evident in the light-on/light-off transition
(Fig. 1A), an observation that is particularly interesting considering
that the visual system is also highly infected ,. However, it
is important to mention that this effect is also clearly detected in
the ‘‘natural’’ activity peak occurring during the last hours of the
photophase (Fig. 1B), which is under circadian control ,,
indicating that a similar effect is likely to occur in nature.
Figure 1. Locomotor activity of control (blue line) and infected (red line) Ae. aegypti females under LD 12:12. Lines represent the hourly
mean activity (+/2 SEM) of control and infected females in the four experiments, normalized to the peak of activity of each respective control. The
grey shadow represents the dark phase. ZT is the Zeitgeber Time. Light turns on at ZT 0 and turns off at ZT 12. Panel (A) shows a full LD cycle while
panel (B) shows only the photophase.
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Other authors have already observed alterations in Aedes
behavior induced by virus infection. Grimstad et al  studied
the feeding behavior of Ae. triseriatus females infected with La
Crosse virus and reported that infected mosquitoes tend to probe
more and engorge less than uninfected females. These results are
in accordance with those obtained by Platt et al , who showed
that the time required for feeding by DENV-3 infected mosquitoes
was significantly longer than that required by uninfected
mosquitoes. In contrast, Putnam and Scott  observed that
DENV-2 infection did not alter Ae. aegypti female blood-feeding
duration and efficiency in an uninfected host. An explanation for
this difference might be that these authors infected mosquitoes
with different Dengue virus (3 and 2, respectively) and that
Putnam and Scott  fed mosquitoes 14 days after an
intrathoracic infection while Platt et al  only observed
significant differences in mosquitoes fed 5, 8 and 11 days after
infection. In our study, we observed locomotor activity differences
in DENV-2 infected mosquitoes 2 to 6 days after intrathoracic
A considerable amount of information is currently available on
the Aedes aegypti immune response to Dengue virus infection –
. Several authors have shown an association between circadian
rhythms and infection/immunity in insects (e.g. –). For
example, Shirasu-Hiza et al  showed that Drosophila infected
with bacterium exhibit disrupted circadian activity rhythms and
that clock gene mutants are more susceptible to infection than
wild-type flies. Also, Lee and Edery  showed that Drosophila’s
ability to fight infections is under circadian control and that flies
are significantly more resistant to bacterium when infected in the
middle of the night than during the day.
It has been shown that several genes from Aedes aegypti are up or
down-regulated upon Dengue virus infection, and in DENV-2
infected mosquitoes at least one orthologue (AAEL012562) of a
Drosophila gene involved in the control of circadian rhythms, Clock,
has its expression nearly doubled after infection . We believe
this variation in a gene probably central to the control of mosquito
circadian rhythms could also contribute to the observed changes in
activity behavior and we are currently investigating whether
Dengue virus infection alters the circadian expression patterns of
other clock genes .
We are aware that our study suffers from possible caveats. For
example, we see a large variation in behavioral effects of Dengue
infection between experiments that we cannot explain at the
moment. Nevertheless, our study shows that Dengue infection
increases mosquito locomotor activity. Changes in vector behavior
caused by infection can have potential epidemiological implica-
tions. Our results encourage further studies to assess whether
increased locomotor activity could have an impact on virus
transmission dynamics and Dengue epidemiology.
We thank Marcelo Quintela and Kathleen Gonc ¸alves for technical
assistance and Luciano Moreira, Pedro Oliveira and Denise Valle for
reading earlier versions of the manuscript.
Conceived and designed the experiments: TC RB MS AP. Performed the
experiments: TC RB. Analyzed the data: TC RB PL AP. Contributed
reagents/materials/analysis tools: MC RO. Wrote the paper: TC RB PL
RO MS AP.
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