Journal of Medical Entomology, 53(2), 2016, 466–469

doi: 10.1093/jme/tjv188
Advance Access Publication Date: 30 November 2015
Short Communication Short Communication

Interspecific Interactions Between Adult Aedes albopictus

and Culex quinquefasciatus (Diptera: Culicidae)
Silvano Daniels, Nnaemeka F. Ezeakacha, and Donald A. Yee1
1
Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39460 (sd447@exeter.ac.uk;
ezeakacha.fn@gmail.com; donald.yee@usm.edu), and 1Corresponding author, e-mail: donald.yee@usm.edu

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Received 20 August 2015; Accepted 4 November 2015

Abstract
Single and mixed species densities of adult Aedes albopictus and Culex quinquefasciatus were manipulated to
determine if different combinations affected their egg laying preference or mortality rates. Oviposition was
measured in environments that contained containers of different surface areas (small cups vs. larger bowls),
and the number of eggs (Aedes) and egg rafts or larvae (Culex) deposited by each species was examined with
respect to intra- and interspecific density treatment levels. Mixed species densities did not have an effect on
survivorship, but single species densities did affect longevity, with higher densities leading to shorter life spans
in Cx. quinquefasciatus: Cx. quinquefasciatus lived longer than Ae. albopictus overall. There was no significant
effect of density combinations on oviposition patterns in either species, but Cx. quinquefasciatus laid more rafts
in bowls compared with cups. There is little evidence of adult interactions between these species; however,
future experimental work is necessary to more fully characterize the possible effects of adult interactions on
these species.

Key words: Aedes, Culex, interspecific, oviposition, survival

An important variable affecting mosquito vector populations in
nature is larval competition. There has been extensive research con-
ducted on competition between different species of mosquito larvae
(Sunahara and Mogi 1997, Reiskind and Lounibos 2009, Burke
et al. 2010, Alto et al. 2015), with findings often yielding situations
of strong asymmetry (Juliano 2010) and context dependence
(Costanzo et al. 2005). Competition can affect specific life-history
traits, which in turn may affect viral transmission (Alto et al. 2005,
2008, 2015). Although there has been an emphasis on larval interac-
tions, few investigations exist documenting interactions at the adult
stage. For example, Rey and O’Connell (2014) determined that
interactions between adult Aedes aegypti and Aedes albopictus
affected egg-laying patterns at adult oviposition sites. Adult interac-
tions are less likely to occur compared with larval interactions, as
larvae are often limited by nutrients. Nutrient limitation, which is
often the basis for larval competition, likely is less important for
fully formed adults. Larvae are confined to their shared aquatic hab-
itats, thus increasing the opportunities for interactions between indi-
viduals, but interactions between adults may still be significant and
may help to better understand populations of vectors in nature,
especially if such interactions affect densities around shared oviposi-
tion sites.
The goal of this research was to test the hypothesis that adult
female Culex quinquefasciatus (L.) and Aedes albopictus (Skuse)
interact in ways that may affect aspects of their ecology. Aedes albo-
pictus and Cx. quinquefasciatus are abundant in artificial containers
in the south (Yee 2008), and are abundant in tire habitats (Qualls
and Mullen 2006; Yee et al. 2012, 2015), where they often are the
dominant species (Yee et al. 2012) and where larvae are known to
compete (Allgood and Yee 2014). Both species are also invasive,
with the potential to negatively affect other species (Juliano 2010).
Aedes albopictus appeared in the United States in 1995 (Hawley
et al. 1987), whereas Cx. quinquefasciatus likely arrived from
Africa over a century ago (Ross 1964, Vinogradova, 2000). Aedes
albopictus routinely lays eggs on the walls of artificial vessels and
can spread a single batch among multiple sites (Hawley 1988); ovi-
position is increased by detritus infusions (e.g., grass and leaves;
Obenauer et al. 2010). Conversely, Cx. quinquefasciatus lay their
eggs on the surface of water with high surface areas (Subra 1981,
Millar et al. 1994) and produce eggs in rafts (Wachira et al. 2010),
and thus all their eggs are laid in a single environment at one time.
We investigated if oviposition choices were affected by intra- or
interspecific interactions in the same environment that contained
aquatic habitats of different sizes, and if these interactions could
affect survival patterns. We predicted that egg-laying patterns would
be altered under mixed species combinations, especially if adult
interactions took place at or near oviposition sites. We also pre-
dicted that longevity would be shorter in the mixed species cages
owing to increased activity as a result of the presence of the second
species. It was also predicted that higher density levels, regardless of
species, would increase the mortality rate of both species via a simi-
lar mechanism.

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Journal of Medical Entomology, 2016, Vol. 53, No. 2
Materials and Methods
Oviposition Experiment
Adults (F1) of each species were generated from laboratory colonies
established from larvae or eggs collected in Hattiesburg, Mississippi.
Eggs were produced from adults that were blood-fed using Japanese
quail, Coturnix japonica (IACUC #11092207). Larvae were reared
in pans filled with reverse osmosis (RO) water and fed a diet of a 1:1
ratio by weight of lactalbumin-yeast powder (0.15 g on days 1, 4,
and every other day thereafter) in a controlled environment incuba-
tor until pupation. Pupae were isolated and transferred into a colony
cage to develop into adults.
For the oviposition experiment, adult female mosquitoes were
blood-fed as stated above. Adult female mosquitoes were left in colony
cages with males for about a week to ensure mating. Afterwards,
females were blood-fed as stated previously. After approximately
48 h, adult females were anesthetized using CO2 and separated into
plastic cages (30 by 25 by 28 cm) with mesh panels for ventilation.
Our densities were approximately 14 times higher as in a study testing
interactions between adults of two species of Aedes (Rey and
O’Connell 2014). We placed a plastic bowl (41 cm in diameter by
5 cm in depth) and cup (28 cm in diameter by 4 cm in depth) at oppo-
site ends of each cage. Bowls and cups were lined with brown paper
and filled with 300 or 340 ml of infusion water, respectively. Infusion
water was created using plant detritus and dead crickets soaked in tap
water for one week to provide an oviposition cue for gravid female
mosquitoes. Sugar pads (cotton soaked in 10% sucrose solution) were
provided as a source of nutrition for adults. Cages were placed in an
incubator set to 27 C, 80% humidity, and a photoperiod of 14:10
(L:D) h. To test the effect of intra- and interspecific adult interactions,
the following species combinations were used: 4:0, 0:4, 8:0, 0:8, 4:4,
8:8, 4:8, and 8:4 (Ae. albopictus:Cx. quinquefasciatus). All combina-
tions were replicated four times, except 0:4 and 4:0, which were repli-
cated five times. After 48 h, the number of eggs laid (Aedes) on paper
or rafts on the surface (Culex) were recorded. Because some rafts
hatched and disassociated before 48 h, we also hatched, counted, and
analyzed the total number of Culex larvae in each container.
Survivorship Experiment
Virgin adult female Ae. albopictus and Cx. quinquefasciatus were
placed into intra- and interspecific combinations (Aedes:Culex: 1:0,
0:1, 2:0, 0:2, 2:1, and 1:2) with 15 replicates of each combination.
Females were separated from males after they eclosed from pans and
were not given a bloodmeal. Flight cages (30 by 25 by 28 cm) were
provisioned with a source of RO water to prevent dehydration, but
without sugar. Cages were kept in an environmental chamber set at
27 C, 80% humidity, and a photoperiod of 14:10 (L:D) h. Daily
adult mortality was recorded for 14 d. There were 16 mosquitoes
that escaped during the experiment, which were excluded from
analysis.
Data Analysis
For the oviposition experiment, analysis of variance (ANOVA) was
used to compare the number of Ae. albopictus eggs, Cx. quinquefas-
ciatus egg rafts, and Cx. quinquefasciatus larvae, separately, present
in bowls and cups among the different density treatment levels. For
the survivorship experiment, failure time analysis (PROC
LIFETEST, SAS 2004) was used to test differences in longevity
across the treatments. Specifically, differences between 1) species, 2)
densities, and 3) and their interaction were tested. Based on the out-
comes of those tests, we also examined differences between species
in low (1 adult) and high (2 adults) combinations, between high and
467
Table 1. Results of separate one-way analyses of variance on Ae.
albopictus egg count, Cx. quinquefasciatus egg rafts, and Cx. quin-
quefasciatus larval count across intra- and interspecific adult
densities
Source df SS MS F P-value
Aedes albopictus eggs
Aedes albopictus (AA) 1, 24 0.1150 0.1150 0.9900 0.3323
Culex quinquefasciatus (CX) 2, 24 0.0930 0.0465 0.4010 0.6754
AA 3 CX 2, 24 0.0709 0.0355 0.3060 0.7401
CX egg raft count
AA 2, 24 0.5180 0.2590 0.3180 0.7315
CX 1, 24 0.0011 0.0011 0.0014 0.9706
AA 3 CX 2, 24 2.0130 1.0070 1.2360 0.3129
CX larval count
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AA 1, 24 0.1150 0.1150 0.9900 0.3323
CX 2, 24 0.0930 0.0465 0.4010 0.6754
AA 3 CX 2, 24 0.0709 0.0355 0.3060 0.7401
low intraspecific densities, and across interspecific interactions (1:0
vs. 1:1, vs. 1:2, and 0:1 vs. 1:1 vs. 2:1). Wilcoxon tests were used to
assess significance in all cases (Sokal et al. 1995).
Results
Oviposition Results
In total, 2,167 Ae. albopictus eggs and 63 Cx. quinquefasciatus egg
rafts were laid during the experiment. Based on ANOVA, the num-
ber of Ae. albopictus eggs, Cx. quinquefasciatus rafts, and Cx. quin-
quefasciatus larvae did not significantly differ between intra- and
interspecific treatment levels (Table 1). Overall, Ae. albopictus did
not show a preference for either cup (1,140 eggs, 51.3%) or bowl
(1,027, 48.7%). Culex quinquefasciatus did appear to exhibit a
preference for bowls, with 25 rafts (39.6%) laid in cups and 38
(60.3%) laid in bowls. In total, 6,512 Cx. quinquefasciatus larvae
were hatched and reflected a similar pattern as rafts (28.1% in cups,
71.9% in bowls).
Survivorship
We found that the effects of species (v2 ¼ 119.12, df ¼ 1, P < 0.001;
Fig. 1), density (v2 ¼ 67.58, df ¼ 6, P < 0.001), and their interaction
(v2 ¼ 132.75, df ¼ 9, P < 0.001) were all significant. Female Cx.
quinquefasciatus in low intraspecific densities survived longer than
those in the high density (v2 ¼ 4.41, df ¼ 1, P ¼ 0.034; Fig. 2).
Additional follow-up tests examining low and high combinations
regardless of species (v2 ¼ 1.88, df ¼ 1, P ¼ 0.180), between high
and low intraspecific densities for Ae. albopictus (v2 ¼ 0.15, df ¼ 1,
P ¼ 0.694), and across interspecific densities (Ae. albopictus, 1:0 vs.
1:1, vs. 1:2, v2 ¼ 2.06, df ¼ 2, P ¼ 0.356 and Cx. quinquefasciatus,
0:1 vs. 1:1 vs. 2:1, v2 ¼ 3.32, df ¼ 2, P ¼ 0.201) were all
nonsignificant.
Discussion
We found no support for our hypothesis that interspecific combina-
tions of adult Cx. quinquefasciatus and Ae. albopictus would affect
survival or oviposition patterns under laboratory conditions. Given
the small size of our cages compared with natural conditions and
larger cages used in a recent study (Rey and O’Connell 2014), we
provided more optimal circumstances for adult interactions to mani-
fest. Adults did not appear to interact in any meaningful way, which
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Fig. 1. Survivorship curves for Aedes albopictus (AA) and Culex quinquefasciatus (CQ) (proportion 6 SE). The experiment was run for 14 d, at which time any liv-
ing mosquitoes were considered as censored variables for the analysis.

Fig. 2. Survivorship curves for low (1 adult) and high (2 adults) densities of Culex quinquefasciatus (CQ) (proportion 6 SE). The experiment was run for 14 d, at
which time any living mosquitoes were considered as censored variables for the analysis.

suggests that such interactions may be infrequent or absent under
natural conditions, where adults freely move in and out of oviposi-
tion sites. However, higher densities of adult mosquitoes, which
likely occur around productive container sites, may still produce
limited effects. There are several factors that may decrease the likeli-
hood for interactions between these species. Culex quinquefasciatus
is not container specialist (Siverly 1972) and has existed in North
America for well over 100 yr (Ross 1964), whereas Ae. albopictus is
a container specialist (Hawley 1988) and invaded just 30 yr ago
(Hawley et al. 1987). Thus, there has been little opportunity for
selection to shape the interactions of these species around contain-
ers. In addition, there are differences between species in the timing
of oviposition (afternoon for Ae. albopictus, dusk for Cx.
quinquefasciatus) and the specific location where eggs are placed
that may minimize opportunities for adults of these species to
interact.
Regardless of interspecific interactions, we did find differences in
oviposition preferences between species. Culex quinquefasciatus
have a natural proclivity to oviposit in larger bodies of water with a
high surface area (Millar et al. 1994); the majority of egg rafts and
larvae in this experiment were found in bowls. Female Cx. quinque-
fasciatus also rely on the egg pheromone 6-acetoxy-5-decanolide,
produced as apical droplets on top of egg rafts, to attract gravid
females to oviposit (Clemens 1999). In one replicate that contained
eight adults of each species, eight egg rafts were found in the bowl.
This result illustrates an example of expected Cx. quinquefasciatus
Journal of Medical Entomology, 2016, Vol. 53, No. 2
oviposition patterns in a container with high surface area and per-
haps pheromones. Conversely, Ae. albopictus egg distributions were
approximately equal between cups and bowls. Unlike species that
share oviposition site selection (Rey and O’Connell 2014), a lack of
interspecific interactions could have been owing to differences in
egg-laying methods, with Cx. quinquefasciatus laying rafts on the
water and Ae. albopictus laying on the wall, thereby decreasing
direct competition for oviposition space. Our experiment suggests
that inherent differences between species, including preference for
certain surface areas, egg pheromone production, and egg location
preferences may supersede the presence of other mosquito species,
preventing interference in adults of these species.
Strong differences in survival between species also were found,
with the average life span for Ae. albopictus at 6.33 d, whereas Cx.
quinquefasciatus survived, on average, for 12.41 d (Fig. 1). Aedes albo-
pictus have been shown to have a higher energy demand, which can be
seen in their larvae’s need for frequent feeding (Barrera 1996). Culex
quinquefasciatus are also larger than Ae. albopictus (Allgood and Yee
2014), thus providing them with more stored energy reserves accumu-
lated as larvae. Beyond species differences, intraspecific densities were
most detrimental to survival for Cx. quinquefasciatus (Fig. 2). This
may be related to several factors, including competition for food and
reproduction locations that suit their niche in the environment. This is
an unlikely explanation, given the controlled nature of our experiment;
however, vibrations from mosquito wings may be one explanation for
differences across densities. Wing vibrations, primarily characterized as
a way for males to find conspecific females, can also be used as a gen-
eral means of communication (Roth 1948, Clements 1999). Higher
intraspecific densities may mean a higher persistence in frequency, and
this may signal others to fly more, increasing overall energetic output.
Interactions between females of the same species are also understudied,
but could be another explanation for the density effects we found here.
Other factors that affect survivorship and oviposition,
including oviposition site marking, aggressive behavior by females,
and the presence of other individuals (Rey and O’Connell 2014), only
add on to the multitude of variables that affect mosquito populations.
Future studies that focus on these and other potential adult interac-
tions may lead to better characterization of vector ecology.
Acknowledgments
We thank S. Schelble and W. Glasgow for assistance with these projects and
three anonymous reviewers for helpful suggestions on this manuscript. This
work was supported by the Department of Biological Sciences at the
University of Southern Mississippi, The Honor’s College, and by grants to D.
A. Yee from the National Institutes of Health (R15[AI]-92404-01A1) and
D.A. Yee from the National Science Foundation (DB I0923063).
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