# Both infected and uninfected mosquitoes are attracted toward malaria infected birds

Stéphane Cornet, Antoine Nicot, Ana R Rivero, Sylvain Gandon

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Stéphane Cornet, Antoine Nicot, Ana R Rivero, Sylvain Gandon. Both infected and uninfected mosquitoes are attracted toward malaria infected birds. Malaria Journal, 2013, 12 (1), 10.1186/1475-2875-12-179. doi:10.1186/1475-2875-12-179

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Both infected and uninfected mosquitoes are
attracted toward malaria infected birds

Stéphane Cornet<fnref n="1" />,<fnref n="2" />*, Antoine Nicot<fnref n="1" />,<fnref n="2" />, Ana Rivero<fnref n="2" /> and Sylvain Gandon<fnref n="1" />

**Abstract**

**Background:** The biting behaviour of mosquitoes is crucial for the transmission of malaria parasites. This study focuses on the feeding behaviour of *Culex pipiens* mosquitoes with regard to the infection status by the avian malaria parasite *Plasmodium relictum* (lineage SGS1).

**Methods:** Uninfected and sporozoite-infected mosquitoes were provided with a choice between an uninfected bird and a bird undergoing a chronic *P. relictum* infection. Mosquito choice is assessed by microsatellite typing of the ingested blood.

**Results:** Chronically infected birds are more attractive to mosquitoes. This choice is not altered by the infection status of the mosquitoes: both infected and uninfected mosquitoes have similar host choice behaviours and are more attracted towards infected birds.

**Conclusions:** These results support some, but not all predictions derived from the hypothesis that malaria parasites can manipulate the behaviour of their mosquito vectors to enhance their transmission. The possible mechanisms driving this manipulation, the evolutionary dynamics leading to the modification of the biting behaviour of mosquitoes by *Plasmodium sp.* as well as the implications for malaria epidemiology are discussed.

**Keywords:** *Plasmodium relictum*, Malaria, Vector behaviour, Attractiveness

**Background**

The manipulation of host phenotypes by parasites, aiming at enhancing parasite transmission, is a wide-spread phenomenon having far-reaching ecological and epidemiological implications [1,2]. In vector-borne diseases the completion of pathogen transmission requires: (i) uninfected vectors to bite infected hosts; and, (ii) infected vectors to bite uninfected hosts. There is growing evidence that parasites may manipulate their vectors at both of these stages of its life cycle.

Parasites have developed strategies to modify the behaviour of uninfected vectors. There is, indeed, growing evidence that uninfected vectors show a preference for feeding on infected hosts [3-7]. Such a preference has been shown in uninfected mosquitoes faced with both

human [8] and avian [9] *Plasmodium sp.* infections. Using the avian malaria parasite *Plasmodium relictum* (lineage SGS1), Cornet *et al*. have recently shown that malaria infection increases the attractiveness of domestic canaries to uninfected *Culex pipiens* mosquitoes, one of the main natural vectors of this parasite [9]. Despite the fact that taking an infected blood meal is costly for the vector (it results in a 30% reduction of mosquito fecundity, [10]) uninfected mosquitoes showed a clear preference towards biting chronically infected birds [9]. A likely adaptive explanation for this seemingly paradoxical behaviour from the mosquitoes' point of view is that it is the result of parasite manipulation. Although the proximal cause leading to the increased attractiveness of infected birds is as yet unclear, it is likely to involve the modification of key odorant volatiles [4,9].

Infected vectors may also be manipulated by the parasite and in different ways. Vectors infected with transmissible parasite stages of *Plasmodium* [11-18], *Leishmania* [19,20] and dengue virus infection [21] have been shown to

<footnote>* Correspondence: stephan.cornet@gmail.com</footnote>

<footnote n="1">¹Centre d'Ecologie Fonctionnelle et Evolutive (CEFE), UMR CNRS 5175, Montpellier, France</footnote>

<footnote n="2">²Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), UMR CNRS 5290-IRD 224-UM1-UM2, Institut de Recherche pour le Développement, 911 av. Agropolis, Montpellier 34394, France</footnote>
exhibit increased attraction to hosts, higher host probing/
biting rates and/or longer blood meals. In addition,
infected vectors are also more likely to feed on multiple
hosts [20,22]. A high-bite strategy, which is probably
achieved through modifications in the composition of the
saliva that hamper the blood meal process [12,23,24], can
be particularly advantageous for parasite transmission,
specially if some of the extra bites take place in new, unin-
fected hosts. Another way to increase transmission may
involve the modification of the host choice behaviour of
the infected vector. Parasite transmission could be in-
creased if infected vectors avoid within-host competition
by feeding preferentially on uninfected hosts. Within-host
competition can take different forms [25]. Parasites may
compete directly for host resources, such as a particular
cell or tissue, or a key limiting nutrient in short supply
(exploitation competition). In addition, parasites may
compete indirectly via the non-specific arm of the host's
immune system (immune-mediated apparent competi-
tion). Teasing apart which mechanism is at play is not an
easy task, but there is evidence from malaria that both of
these mechanisms may be acting simultaneously [26-31].
The study of the host-choice behaviour of infected vectors
remains, however, poorly studied. Some infected vectors
of plant pathogens have been shown to be manipulated to-
wards uninfected plants [5,6]. To date, no studies have
ever looked at the host choice behaviour of malaria-
infected mosquitoes.

The present study investigates the biting behaviour of
both uninfected and sporozoite-infected *Cx. pipiens*
mosquitoes when given the choice between birds that
are uninfected or in chronic *P. relictum* infection. The
number of mosquitoes that fed on each host has been
quantified using genetic (microsatellite) analyses of the
blood meal [9]. This system gives a more biologically
relevant measure of host choice as it captures the whole
behavioural sequence from the detection of the odour to
the decision to bite.

This experimental set up allowed testing three predic-
tions deriving from the manipulation hypothesis. First,
uninfected mosquitoes are expected to feed preferen-
tially on the infected birds [9]. Second if, as discussed
above, *P. relictum* can manipulate the biting rate of the
infected mosquitoes, one may expect the infected mos-
quitoes to have a higher probability of feeding and a
higher probability of multiple-host biting than unin-
fected mosquitoes. Third, if *P. relictum* can manipulate
the host-choice of the infected mosquitoes, one may
expect the vectors to avoid within-host competition by
preferentially biting the uninfected hosts. Note that this
may generate a conflict of interest between the parasite
in the bird (trying the attract mosquitoes) and the para-
site in the mosquito (trying to avoid infected birds). The
experiment is designed to study how this conflict is

resolved and to obtain a more precise description of
malaria transmission. As discussed below, a better know-
ledge of the biting behaviour of the vector can have im-
portant consequences for the epidemiological dynamics
of vector-borne diseases [32,33].

Methods

Malaria parasites

*Plasmodium relictum* (lineage SGS1) is the aetiological agent of the most prevalent form of avian malaria in Europe [34] and is highly prevalent in wild passerines [35-37]. This generalist *P. relictum* parasite lineage was originally isolated from wild house sparrows caught in the region of Dijon (France) in 2009 [38] and maintained in the laboratory via subsequent passages to naïve canaries by intraperitoneal injection or by completing the parasite cycle through mosquitoes. Mosquitoes of the *Cx. pipiens* complex are the main vectors of *P. relictum* in the field [37].

Infected and uninfected mosquitoes

Experiments were conducted with a laboratory strain of
*Cx. pipiens quinquefasciatus* (SLAB) [39]. Mosquitoes
were reared under standard conditions [40]. Infected
and uninfected mosquitoes for the experiment were
obtained in the following way. Eight cages (dimensions
L40 × W30 × H30 cm), each containing 150 female (six
to seven days old) mosquitoes were set up. Half of these
cages were provided with an infected canary, the other
half with an uninfected one. Infected birds had been in-
oculated with the parasite 12 days previously following
standard laboratory procedures, and were thus at the
acute stage of the infection. Previous work has shown
that this protocol ensures that >90% of the mosquitoes
become infected [40]. Unfed mosquitoes were discarded.
Four days after the infected or uninfected blood meal
(day 4 pbm), and until the beginning of the behavioural
assay, mosquito cages were provided with a water-filled
plastic tray to allow females to lay their eggs. On day 7
pbm, a subsample of ten mosquitoes were haphazardly
collected from each of the infected cages and dissected
to verify the presence of oocysts in the midgut [40].
Only the three of the four infected cages that reached a
prevalence of infection of 100% were kept for the experi-
ment. A similar sample of mosquitoes was collected
from the uninfected cages to verify the absence of
parasite.

Once mature (eight to nine days pbm), oocysts burst
and release sporozoites into the mosquito body cavity
that rapidly colonize the salivary glands within hours
[41]. Parasite sporozoites can be detected within the
glands starting from day 10 pbm (A Nicot, unpublished).
The timing of the behavioural assay was set to coincide
with the peak of sporozoite presence in the salivary
glands of infected mosquitoes (12–14 days pbm). Given that *P. relictum* sporogony is asynchronous, some midgut oocysts may, however, still be developing at this stage (see [42]). Four days before the behavioural assays, mosquitoes were marked using small amount of either pink or yellow fluorescent powder (RadGlo_JST, Radiant Color NV, Houthalen, Belgium) applied as a dust storm [40]. The two colours were used in rotation to mark uninfected and infected mosquitoes.

## Infected and uninfected birds

One year-old canaries (*Serinus canaria*) were used in this experiment. A small amount (*ca.* 20–30 µL) of blood was collected from the brachial vein and used for molecular sexing [43] and genotyping at the microsatellite Cuμ28 locus [9,44]. At the same time, the birds were verified to be free from any haemosporidian infections [45]. Birds were then assorted into ten different same-sex pairs making sure that birds within a pair had different microsatellite profiles at the Cuμ28 locus [9]. Within each pair, one bird was randomly chosen to be infected. Infections were carried out using an intraperitoneal injection of *ca.* 50–100 µL of blood from the infected canary stock [9]. The success of the infection was verified 11 days post-infection (dpi) using thin blood smears stained with Giemsa.

## Behavioural assay

The behavioural assay took place 53–55 dpi, to coincide with the chronic stage of the bird infection. On the same day, but prior to the assay, a small amount (*ca.* 20–30 µL) of blood was taken from the brachial vein of each of the birds to measure parasitaemia and haematocrit level. Parasitaemia was established using previously published qPCR procedures [9]. Haematocrit level was expressed as the packed cell volume (PCV) volume of red blood cells per total volume of blood in the capillary after centrifuging blood for 5 min at 10,000 rpm [9].

To minimize host defensive behaviours that may alter the mosquito feeding process during the assay [46], birds were immobilized in a specially designed PVC tube that rendered their legs accessible to the mosquitoes while protecting the rest of the body from the bites [9]. Each bird pair was placed inside a cage (dimensions L80 x W30 x H30 cm) with 40 uninfected and 40 sporozoite-infected female mosquitoes for two hours (from 6 to 8 pm). Each batch of 40 infected and 40 uninfected mosquitoes contained an equal proportion of mosquitoes from the three infected and four uninfected cages. To avoid interference, each pair of birds was assessed in a separate controlled temperature room (four different CT rooms, temperature: 25 ± 1°C, relative humidity: 73 ± 3%). The experiment was spread over three consecutive evenings. After each run, all mosquitoes were taken out

of the cage and stored at -80°C for microsatellite analyses. Microsatellite analyses of the blood fed mosquitoes were carried out using a previously published protocol [9]. Mosquitoes were sorted as unfed when no microsatellite signal was detected.

## Statistics

The analyses were carried out using generalized linear mixed models (glmer, package lme4) available in the R statistical package (v. 2.15.2). The binomially distributed response variables were: the proportion of mosquitoes that took a blood meal (blood feeding success), the proportion of mosquitoes that fed on both birds (multihost biting), and the proportion of mosquitoes that bit the infected bird relative to the total number of blood-fed mosquitoes (multihost feeders were eliminated from this analysis). Models were fitted by specifying mosquito infection treatment (infected, uninfected) as a fixed effect and the bird pair identity as a random effect [47]. In addition, the effect of Δ haematocrit (the difference in haematocrit between the infected and the uninfected bird) and blood parasitaemia on vector-feeding preference were investigated [9]. Maximal models were simplified by sequentially eliminating non-significant terms and interactions (*p* > 0.05) and comparing the change in deviance with and without the term using a χ² distribution. The effect of *P. relictum* infection on bird attractiveness was confirmed by using a replicated *G*-test of goodness of fit [9,48]. The observed attractiveness of the infected bird (proportion of mosquitoes biting the infected bird) was tested against the predicted no-choice value of *p* = 0.5 (half of the mosquitoes bite each of the birds).

## Ethical statement

Animal experiments were carried out in strict accordance with the “National Charter on the Ethics of Animal Experimentation” of the French Government, and all efforts were made to minimize suffering. Experiments were approved by the Ethical Committee for Animal Experimentation established by the authors’ institution (CNRS) under the auspices of the French Ministry of Education and Research (permit number CEEA-LR-1051).

## Results

The status of birds was confirmed by qPCR assays: control birds were parasite-free and experimentally infected birds were all positive for *P. relictum* infection. Since birds harboured chronic (low intensity) infections, the infection status did not affect bird haematocrit (mean ± se: uninfected 0.43 ± 0.02, infected 0.40 ± 0.03, $F_{1, 18} = 0.93, p = 0.3485$) and overall, within pairs, uninfected
and infected birds had similar haematocrit values (one-sample *t*-test *t*<sub>1,9</sub> = -1.16, *p* = 0.2763).

The majority of mosquitoes (> 90%) took a blood meal and the success of engorgement did not depend on whether mosquitoes were infected (mean ± se: 0.945 ± 0.010) or uninfected (0.942 ± 0.009, $\chi_1^2$ = 0.05, *p* = 0.8261, Figure 1A). The proportion of mosquitoes that fed on both birds was low and did not differ between infected (mean ± se: 0.061 ± 0.019) and uninfected mosquitoes (0.062 ± 0.016, $\chi_1^2$ = 0.007, *p* = 0.9424, Figure 1B).

<figure><img src="image_2.png" /></figure>

The global GLMM highlighted that the infection status of mosquitoes did not affect the feeding preference ($\chi_1^2$ = 0.29, *p* = 0.5901; Figure 2A). There was neither an

<figure><img src="image_3.png" /></figure>
effect of Δ haematocrit (χ₁² = 0.97, *p* = 0.3252) or bird
parasitaemia (χ₁² = 0.03, *p* = 0.8670) on vector choice.
Both uninfected and sporozoite-infected mosquitoes
preferred feeding on infected birds, however, in this
model, this preference was not significant given the
model intercept did not represent a significant deviation
from 0.5 (*p* = 0.0985). Given the absence of an effect of
mosquito infection on feeding choice, and since there is
still a debate about the correct choice of degrees of free-
dom and the robustness of GLMM analyses, a simplified
GLM was run to investigate bird attractiveness using all
mosquitoes, independently of their infection status, thus
discarding the random term effect (see below).

Infected birds attracted significantly more mosqui-
toes (60.3% of the mosquitoes) than uninfected birds
(χ² = 104.05, *p* < 0.0001; Figure 2A). A replicated *G*-test
of goodness of fit confirmed a statistically significant
departure from the frequency in the absence of choice
(p = 0.5) (total-*G* = 106.67, 10 *df*, *P* < 0.0001; pooled-*G* =
15.16, 1 *df*, *P* < 0.0001), albeit with a significant heterogen-
eity between the replicates (heterogeneity-*G* = 91.50, 9 *df*,
*P* < 0.0001). There was a significant positive effect of Δ
haematocrit on the proportion of mosquitoes biting the
infected bird (Figure 2B; χ² = 11.05, *p* = 0.0009), suggesting
that mosquitoes prefer biting the birds with high haem-
atocrit values. However, bird parasitaemia did not
quantitatively influence the strength of mosquito
feeding behaviour (χ² = 0.25, *p* = 0.6161). Further ana-
lyses confirmed that infected and uninfected mosqui-
toes behaved in the same way: within each bird pair
there was a significant positive covariation in the
choice of infected and uninfected mosquitoes (*r* =
0.767, *F*₁, ₈ = 11.45, *p* = 0.0096; Figure 3).

**Discussion**

A previous experiment has shown that *P. relictum* infec-
tion increases bird attractiveness to mosquitoes: about
60% of uninfected mosquitoes preferred feeding on birds
with a chronic infection (24–26 dpi) compared to unin-
fected controls [9]. In the present study, results are
quantitatively similar, albeit in even older infections (53–
55 dpi). The haematocrit effect found in the previous
paper is also present here: mosquitoes prefer biting the
least anaemic birds. Avian malaria infections can last for
months in their chronic state (S Cornet *et al*., unpublished;
[49]). An increase in the attractiveness of the
birds during this stage may thus enhance considerably
malaria transmission. This result is thus consistent with
the first prediction of the manipulation hypothesis.

In contrast, the second of the predictions, concerning
the probability of biting of infected mosquitoes does not
seem to be supported by the data. There was no differ-
ence between uninfected and sporozoite-infected *Cx.
pipiens* in either the blood feeding success (Figure 1A)

or the rate of multihost biting (Figure 1B). This con-
trasts with earlier studies reporting an increased attraction
to hosts [17,18] and altered biting rate of *Plasmodium*
*sp.*-infected mosquitoes [12-16,22]. Here, however, it was
impossible to control for the duration and the volume of
the blood meals. More detailed behavioural studies should
be carried out on avian malaria to investigate the possibil-
ity that *P. relictum* manipulates these components of the
dynamics of the biting behaviour of infected mosquitoes.

Finally, contrary to the third and final prediction of
the manipulation hypothesis, uninfected and sporozoite-
infected mosquitoes behaved in a similar way (Figure 3):
they were both preferentially attracted by chronically
infected birds (Figure 2). Biting an infected host was
predicted to be costly for the malaria parasite within the
mosquito because of within-host competition with resi-
dent parasites and/or immune mediated apparent com-
petition [25]. In several plant pathogen systems, infected
vectors are indeed preferentially attracted toward unin-
fected plants [5,6]. However, the results show that, in
the avian malaria system, what is driving the host-choice
of infected mosquitoes is the malaria parasite within the
bird, not the malaria parasite within the mosquito. In
this experiment, the parasite in the bird and in the mos-
quito came from the same parasite stock (lineage SGS1),
differing only by one serial passage. This brings up the
question of whether the results would have been

<figure><img src="image_4.png" /><figcaption>**Figure 3** Covariation between the proportion of uninfected vs. sporozoite-infected mosquitoes biting the bird infected with *Plasmodium relictum*. The significant positive relationship (dash line) highlights that, independently of the intrinsic attractiveness of the infected bird, infected and uninfected mosquitoes had a similar host-choice preference. The dotted lines represent the proportion in the absence of choice (*p* = 0.5).</figcaption></figure>
significant if two different parasite stocks, or parasite
lineages, had been used to infect the birds and the mos-
quitoes. The answer is likely to be no, because costs for
the mosquito-inhabiting parasite of biting an already
infected host are maximal when the bird-residing para-
site is a relative. An analogous problem is found in
parasitoid wasps that encounter hosts that have been
already parasitized, either by itself or by a different
female. In these circumstances, females have been
shown to avoid self-superparasitism and to prefer hosts
parasitized by conspecifics to avoid the costs of sibling
competition [50].

What is driving the bias in the host-choice behav-
iour in mosquitoes? Plant-pathogen studies have gone
very far in deciphering the underlying cues used by
the vectors to choose between infected and uninfected
plants. Some of these cues involve the colour, the
quality and the odours of the plants [4-6]. For malaria,
the vectors often bite in the dark and the choice is
probably not guided by visual cues. The quality of the
blood meal is likely to play a role on this choice: mos-
quitoes are consistently attracted towards birds with a
high haematocrit (see also [9]). High haematocrit cor-
relates with a higher protein content [51] which may,
in turn, correlate with a higher mosquito fecundity or
longevity although, no study to date has investigated
the correlation between anaemia in the host and mos-
quito fitness. Yet, whether and, if so, how do mosqui-
toes evaluate the haematocrit of the birds is an open
question. Given that many diseases are associated with
changes in host odour profile [52], it is likely that
odour volatiles provide a lot of information to the
vectors. Body odours emanating from the gland secre-
tions and the skin microflora play an important role
in mosquito attraction in humans [18,53,54]. In the *P.
relictum* – *Cx. pipiens* – *S. canaria* system, the quantity
and the composition of these volatiles may provide in-
formation to the mosquitoes about the infection status
and haematocrit levels of the bird. A bird-derived odor-
ant molecule, the nonanal, has recently been identified
to be a strong attractant for *Culex* mosquitoes [55] and
could potentially be a key molecule in this system.

Understanding the proximal cues of the attraction may
help understand the evolution of the mosquito manipu-
lation by the malaria parasite. For example, bird infec-
tion may act on the quantity but not on the composition
of the odour. In this case, the increased preference for
infected birds would be triggered by the exaggeration of
a pre-existing cue or a cue that is already used by mos-
quitoes to locate hosts [4]. Evolutionary speaking, such
deceptive signals are a good way to prevent the evolu-
tion of resistance against the attractant in the vector
population, because, for both infected and uninfected
mosquitoes, the fitness costs of ignoring such signals are

large (no blood meal means no reproduction). This
shows that the next experimental step in the study of
mosquito behaviour involves deciphering the odours
emitted by the birds and relating these to mosquito
choice.

Mosquito choice may have a huge impact on the
epidemiology of malaria transmission. In particular,
Kingsolver [32] showed that the higher attractiveness of
infected hosts increases the basic reproductive number,
*R₀*, of malaria. In other words, the effect reported in the
present study could increase the initial spread of the epi-
demic. One interesting situation may take place when
parasite prevalence is high. In this case, increasing the
attractiveness of infected hosts may protect uninfected
hosts, eventually decreasing the incidence of malaria in
the vertebrate population. In this particular situation,
and contrary to the adaptive hypothesis presented
throughout this paper, a preference for infected hosts
may not be adaptive for the malaria parasite. To investi-
gate the adaptive nature of these phenotypic alterations
an evolutionary model is currently being developed
where the choice between infected and uninfected hosts
will be allowed to evolve under different scenarios de-
pending on who is controlling the phenotype: the vector,
the parasite in the host or the parasite in the infected
vector. This may help understand the evolution of
malaria but also the transmission of many other vector-
borne diseases.

Conclusion

This study is the first to investigate the combined effect
of infection by malaria parasites on host attractiveness
and vector feeding preference using a behavioural choice
experiment over the whole *P. relictum* life cycle. Both
sporozoite-infected and uninfected mosquitoes have
similar biting behaviours and are more attracted towards
birds in the chronic infection stage. These results support
some, but not all predictions derived from the hypothesis
that malaria parasites can manipulate the behaviour of
their mosquito vectors to enhance their transmission.
Although the importance of manipulation by malaria
parasites for disease transmission in the field remains to
be confirmed [56], the avian malaria system provides an
unparalleled opportunity to study these questions in
depth. The results obtained using this animal model,
which associates a natural vector-*Plasmodium* sp. combin-
ation, will help improve our understanding of malaria
epidemiology.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SC, AR and SG conceived and designed the experiment. SC performed the experiment and analysed the data. AN performed the molecular analyses, SC,
AR and SG wrote the paper. All authors read and approved the final manuscript.

## Acknowledgements

We are grateful to P Perret for his help with the birds. The work was funded by the CNRS and the ERC Starting Grant 243054 EVOLEPID to SG.

Received: 17 April 2013 Accepted: 27 May 2013

Published: 3 June 2013

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doi:10.1186/1475-2875-12-179

Cite this article as: Cornet et al.: Both infected and uninfected mosquitoes are attracted toward malaria infected birds. *Malaria Journal*
2013 12:179.

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