Potential evidence of parasite avoidance in an avian malarial vector

Animal Behaviour 84 (2012) 539e545

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Potential evidence of parasite avoidance in an avian malarial vector Fabrice Lalubin a, Pierre Bize a, Juan van Rooyen a, Philippe Christe a,1, Olivier Glaizot b, *,1 a b

Department of Ecology and Evolution, Le Biophore, University of Lausanne, Lausanne, Switzerland Museum of Zoology, Lausanne, Switzerland

a r t i c l e i n f o Article history: Received 1 November 2011 Initial acceptance 24 January 2012 Final acceptance 24 May 2012 Available online 4 July 2012 MS. number: 11-00885R Keywords: avian malaria Culex pipiens dual-choice olfactometer great tit host choice mosquito Parus major vector-borne disease

Epidemiological studies of malaria or other vector-transmitted diseases often consider vectors as passive actors in the complex life cycle of the parasites, assuming that vector populations are homogeneous and vertebrate hosts are equally susceptible to being infected during their lifetime. However, some studies based on both human and rodent malaria systems found that mosquito vectors preferentially selected infected vertebrate hosts. This subject has been scarcely investigated in avian malaria models and even less in wild animals using natural hosteparasite associations. We investigated whether the malaria infection status of wild great tits, Parus major, played a role in host selection by the mosquito vector Culex pipiens. Pairs of infected and uninfected birds were tested in a dual-choice olfactometer to assess their attractiveness to the mosquitoes. Plasmodium-infected birds attracted significantly fewer mosquitoes than the uninfected ones, which suggest that avian malaria parasites alter hosts’ odours involved in vector orientation. Reaction time of the mosquitoes, that is, the time taken to select a host, and activation of mosquitoes, defined as the proportion of individuals flying towards one of the hosts, were not affected by the bird’s infection status. The importance of these behavioural responses for the vector is discussed in light of recent advances in related or similar model systems. Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Parasites are ubiquitous organisms and by definition impose fitness costs on their hosts. As a consequence, hosts have evolved a range of antiparasite defences. Together with the immune system (Wakelin 1996), behavioural responses are the most efficient defences that hosts have evolved to fend off the detrimental effects of parasites. Detecting and avoiding parasites is the first line of defence that has been proven efficient at curbing parasite spread. For example, birds have been found to avoid nest sites infected by fleas (Christe et al. 1994) and herbivores such as antelope (Ezenwa 2004) or kangaroos (Garnick et al. 2010) are known to select grass patches for grazing so as to minimize the risk of being infected by gastrointestinal parasites associated with faecal contamination. Hygienic behaviour (Arathi et al. 2006) and the incorporation of nest material that contains substances with antiparasite properties are other behavioural adaptations that animals have evolved to help prevent disease (Petit et al. 2002; Christe et al. 2003; Hart 2005; Castella et al. 2008). Behavioural defences against parasitism have the advantage of providing fast responses to environmental modifications. For example, Hawaiian birds have modified their behaviour in response * Correspondence: O. Glaizot, Museum of Zoology, Place de la Riponne 6, 1014 Lausanne, Switzerland. E-mail address: [email protected] (O. Glaizot). 1 These two authors are the senior authors of this paper.

to selection pressure imposed by the introduction of mosquito vectors and malaria parasites on the islands (van Riper III et al. 1986). It was shown that in a relatively short period of time some bird species had modified their sleeping postures to avoid being bitten by mosquitoes and had changed their daily movements between foraging and sleeping areas in order to minimize their temporal contact with malarial vectors (van Riper III et al. 1986).  nas (2005), belong to the genus Malaria parasites, sensu Valkiu Plasmodium (Apicomplexa: Haemosporidae) and represent a highly diversified, monophyletic group of blood protozoan parasites that can be divided into two well-supported clades, one containing parasites of mammals and the other parasites of reptiles and birds (Bensch et al. 2004; Martinsen et al. 2008; Witsenburg et al. 2012). These parasites exploit a large spectrum of host species that have a broad range of ecological niches, resulting in a world-wide distribution (Garnham 1966). Besides the requirement of a suitable vertebrate host species to multiply asexually, the Plasmodium parasite’s life cycle involves haematophagous dipteran insects for both parasite transmission and sexual reproduction (Garnham 1966). Mammal-specific Plasmodium parasites are well known and have evolved to specialize within Anopheles mosquitoes (Garnham 1966; Martinsen et al. 2008). On the other hand, identification of vectors and their role in the epidemiology of avian malaria in the wild is poorly known. Some mosquito species are competent vectors

0003-3472/$38.00 Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anbehav.2012.06.004

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in laboratory conditions (Huff 1965) but their vectorial capacity (i.e. the efficiency with which the vector transmits the parasite in natural conditions) has not been demonstrated or is based on a PCR detection of the parasite in the mosquito’s body (Massey et al. 2007; Ishtiaq et al. 2008; Ejiri et al. 2009; Glaizot et al. 2012). However, only the presence of the infective stage of the parasite (sporozoites in the salivary glands) clearly demonstrates the competence and vectorial capacity of a given species (LaPointe et al. 2012). Epidemiological studies or models of malarial disease first considered vectors as passive parasite transmitters (MacDonald 1957; Garrett-Jones 1964), with each host having the same probability of being infected (but see Kingsolver 1987; Kelly & Thompson 2000; Smith et al. 2007). However, several studies have challenged this assumption and have shown that hosts are not equally attractive to vectors (Kelly 2001). First, vectors may preferentially select to feed on a given species (Lord & Day 2000; Williams et al. 2003; Lefèvre et al. 2009; Simpson et al. 2009), depending on the diversity and the relative abundance of available host species (Lyimo & Ferguson 2009). Second, individuals of the same host species may represent different levels of attractiveness to the vectors. For instance, humans vary in their intrinsic attractiveness to mosquitoes (Knols et al. 1995; Mukabana et al. 2002; Smallegange et al. 2011) with pregnant women (Lindsay et al. 2000) or beer consumers (Lefèvre et al. 2010) being more attractive than other individuals. By increasing the attractiveness of an intermediate vertebrate host the parasite can improve its transmission to the vector. This can be regarded as a manifestation of the extended phenotype of the parasite (Dawkins 1982) and has been applied to parasites with trophic transmission (Moore 2002; Cezilly et al. 2010) as well as vector-borne diseases (Hurd 2003; Nacher 2005; Adedolapo & Olajumoke 2008). In this way, parasites may gain in terms of reproductive success by manipulating host attractiveness to the vectors. Even though Plasmodium parasites have an interest in keeping their vector alive until transmission to their vertebrate host has occurred, they may not accomplish their cycle without any collateral damage to the vector (Beier 1998; Ferguson & Read 2002; Hurd 2003; Lefèvre & Thomas 2008). Vectors may thus have evolved the ability to discriminate between infected and parasitized hosts to avoid being parasitized (Freier & Friedman 1976; Tomás et al. 2008; Martinez-de la Puente et al. 2009). Nonrandom host-feeding behaviour by the vector may therefore be at the heart of a conflict of interest between Plasmodium parasites and their vectors and may strongly affect the transmission dynamic of the disease (Dye & Hasibeder 1986; Kingsolver 1987; Dye 1992; Smith et al. 2007) as well as vector fitness (Lyimo & Ferguson 2009). This study focused on a temperate avian malaria system involving great tit, Parus major, hosts naturally infected with malarial parasites, Plasmodium spp. (Richner et al. 1995; Oppliger et al. 1996, 1997; Christe et al. 2012) vectored by the ornithophilic mosquito Culex pipiens (Glaizot et al. 2012). The objective was to determine whether Plasmodium infection of great tits affects their attractiveness to wild C. pipiens. Host-seeking vectors were given the opportunity to orient themselves towards their preferred host in a dual-choice olfactometer baited with malaria-infected and uninfected birds. Attractiveness of the bird hosts and activation and reaction time of the mosquitoes were measured. METHODS General Procedure and Study Sites The study took place in western Switzerland. Thirteen wild great tits were mist-netted in two forests situated 30 km apart (Dorigny: 46
310 N, 6
340 E; altitude: 400 m; La Praz: 46
660 N, 6
430 E; altitude: 871 m). Each bird was individually marked with a metallic ring, weighed to the nearest 0.1 g, sexed, aged (1 year old or older

than 1 year based on plumage criteria) and blood sampled for later assessment of their malaria infection status (see below). Birds were then transferred to a mosquito-free animal room (20
C, 50% relative humidity and a 16:8 h light:dark cycle) on the campus of the University of Lausanne in Dorigny, individually housed in aviaries (1  1 m and 2 m high) and provided with ad libitum access to water, mealworms and commercial seed for insectivorous passerines (Peddy seeds, Rolli-pet Tiernahrung GmbH, Hargelsberg, Germany). At the end of the experiment, individuals were blood sampled a second time to reassess their infection status. Culex pipiens mosquitoes were collected as egg rafts in rainfallcollecting containers baited with live yeast and installed within the great tit population site at Dorigny. Containers were checked for egg rafts one to three times per week. Freshly laid eggs were transferred to the laboratory (25
C, 70% relative humidity and a 14:10 h light:dark cycle) and allowed to hatch in plastic trays filled with 1.5 litres of spring water. Larvae were fed ad libitum with commercial fish flakes until pupation; pupae were then isolated in screened cages (30  30  30 cm) for adult emergence. Since C. pipiens are unable to mate in confined spaces (Vinogradova 2000), adult males and females, which had emerged within the same 72 h period, were transferred to bigger screened cages (30  30 cm and 90 cm high) to allow for vertical nuptial flights. A sex ratio of seven males to five females was maintained for 12e16 days (mean  SD ¼ 15  1.3) for mating and fed with a fresh 10% glucose solution renewed every 3 days to prevent fungi formation. Ethical Note Birds used in these experiments were mist-netted between 25 June and 12 July 2010 after the breeding season. They were carefully transferred from the field to the laboratory in muslin bird bags and travel time did not exceed 45 min. Individuals were kept in captivity for 8e28 days (average 16.4 days), including a period of 5e9 days after the last experiment, to ensure that birds were in good health before being released at their capture site. Blood samples of 20 ml were collected into lithium-heparinized microvettes (Microvettes CB 300, Sarstedt, Germany) by puncturing the brachial vein with a sterile needle (Neolus 100, Terumo Europe, Heverlee, Belgium). Bird captures and ringing were performed under licence number F044-0799 of the Swiss Federal Office for the Environment. Experiments with wild great tits were approved by the Veterinary service of the Canton de Vaud, licence number 1730.1. Molecular Identification of Plasmodium spp. from Birds Genomic DNA was extracted from the blood samples using a Qiagen BioSprint 96 workstation following the manufacturer’s protocol (Qiagen, Hilden, Germany). Samples were screened for Plasmodium spp. infection using the nested PCR method refined by Waldenström et al. (2004) from the original protocol made by Bensch et al. (2000). Birds were deemed as malaria infected when a final PCR product of about 525 bp including primers was obtained. Stage of infection (chronic or acute) was not determined with this nonquantitative method. Positive controls were included in all PCR assays. Negative controls (purified PCR-grade water) were included every three to four samples. Reactions were run on a Biometra thermocycler (Biometra, Göttingen, Germany). Olfactometer Set-up Mosquito experiments took place in an olfactometer, consisting of a Y-shaped wind tunnel, as recommended by Besansky et al. (2004) and built with some modifications from the designs of Geier et al.

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(1999) and Cooperband et al. (2008). The wind tunnel was made of transparent Plexiglas (thickness 0.6 cm) and consisted of a box measuring 30  21 cm and 11 cm high connected on the upwind side to two 35 cm long arms of 11 cm in outer diameter and, on the downwind side, to a single arm (42 cm length, 11 cm in outer diameter). The upwind arms were connected to two independent aircircuits with a JBL tube (4 mm in inner diameter). Ambient air was successively forced by an air-pump (WISA 300, ASF Thomas Industries, Puchheim, Germany) into an active charcoal filter to remove any organic contaminants, bubbled through water to maintain constant humidity and pulsed into a plastic chamber (23.6  15.8 cm and 15.8 cm high) equipped with a perch and intended to house the unrestrained bird. Airflow was controlled by a flowmeter (SHO-Rate, Brooks Instrument, Hatfield, PA, U.S.A.; airflow: 8.25 ml/min) before reaching the upwind arms of the set-up. Mosquito release chambers consisted of plastic cylinders (7 cm diameter, 5 cm long) inserted into the downwind arm of the set-up, with a foam plug attached to a string, which was pulled out at the beginning of the experiments, allowing mosquitoes to fly into the wind tunnel. Dual Host Choice Experiment Attractiveness of great tits to C. pipiens mosquitoes was assessed using the Y-shaped wind tunnel allowing the simultaneous presentation of body odours from two birds to a batch of 20 or 40 mated female mosquitoes that had no prior exposure to bird hosts (i.e. experimentally naïve mosquitoes). Before the test was initiated, birds were successively weighed, placed into a chamber, then plunged into darkness and allowed to settle for 5 min. Experimental adult female mosquitoes were sugar deprived for 24 h before testing to increase their motivation to seek a blood meal. Only spring water was provided to avoid desiccation. They were then placed into the release chamber that was plugged into the downwind arm of the olfactometer and female mosquitoes were released into the set-up. They were allowed 15 min to fly upwind following odour cues from the host pair, which was out of sight and out of reach of the mosquitoes. The responding mosquitoes, which entered either of the upwind arms, were prevented from flying back by transparent plastic funnels. After each experiment, the olfactometer and the bird chambers were successively cleaned with commercial detergent, 70% alcohol and distilled water, and then air-dried. Two to six experiments were performed every day for 10 days, each time using a novel batch of experimentally naïve mosquitoes. Experiments began 90 min before the artificial nightfall in the insectary to simulate the conditions when mosquitoes are naturally active. The experimental room was kept at ambient temperature (mean  SD ¼ 25  1
C) and relative humidity (mean  SD ¼ 50  10%). Trials took place in complete darkness and mosquito movements were recorded with an infrared Sony HANDYCAM video camera. Video recordings were then analysed to assess the reaction time of individual mosquitoes. Six of the 13 great tits caught were found to be positive for malaria. We used these six parasitized birds and five uninfected ones to determine whether infection status of the bird affects mosquito host selection. We designed dual-choice experiments based on a random sample of bird pairings consisting of one infected and one uninfected great tit (UI pairing). Thirteen pairs (out of a possible 30) were randomly chosen for the experiments, each pair being tested at least twice by permuting bird placement in the set-up to control for the positional effect. Our design allowed us to minimize the captivity period of the birds throughout the 28 UI experiments performed. Statistical Analysis All statistics were performed using R 2.13.0 (R Development Core Team 2011). Statistical analyses were performed at the

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individual bird level. The attractiveness of a focal bird, infected or not, was defined as a binomial variable by binding (using the cbind function) the number of attracted mosquitoes to the focal bird with the number of attracted mosquitoes to its opponent, summed over all the experiments in which the focal bird was used. The reaction time (s) was defined as the mean time taken by mosquitoes to select a focal bird across all the experiments in which it was used. The degree of activation of mosquitoes confronted with a focal bird pair was defined by binding (cbind function) the number of mosquitoes trapped in the olfactometer (activated) with the number of unactivated mosquitoes (those that stayed in the release chamber) summed over all the experiments performed with this focal bird pair. Data were analysed using generalized linear models (GLM). We analysed the effect of host infection on the attractiveness to the mosquitoes with a quasibinomial error structure and a logit-link function and on mosquito reaction time with a normal error structure. A quasibinomial distribution of the error structure was used to account for overdispersion in our data of attraction. Statistical significance was tested using F statistics (Crawley 2007). We ran additional analyses by sequentially adjusting the models for sex and body mass. Because infection status is partially confounded with the site of origin of the birds (i.e. adult great tits had greater malaria prevalence in Dorigny than La Praz) and with age (i.e. older birds may be more likely to be infected, Knowles et al. 2011), we used experiments challenging three uninfected birds from the forest of Dorigny against three others from La Praz (UU pairings ¼ 6 over 9). We found no significant effect of the site of origin and of age on host attractiveness (all P > 0.20) and mosquito reaction time (all P > 0.20). We analysed the effect of the bird pairing type (UU or UI pairing) on activation by using a GLM model (logit link, quasibinomial error distribution) that was sequentially adjusted for the sum of the body mass of the paired individuals.

RESULTS Attractiveness of bird hosts to mosquitoes was highly significantly affected by bird infection status in the dual-choice experiment (GLM: F1,9 ¼ 18.0, P ¼ 0.002), with uninfected birds attracting more mosquitoes than Plasmodium-infected ones (Fig. 1). Effect of the bird’s infection status on its attractiveness remained significant after the sequential adjustment of the initial model by sex (effect of sex: F1,9 ¼ 6.263, P ¼ 0.037; infection status: F1,8 ¼ 11.475, P ¼ 0.009) and body mass (effect of body mass: F1,9 ¼ 4.391, P ¼ 0.069; infection status: F1,8 ¼ 11.615, P ¼ 0.009). Although sex accounted for a significant part of the variance explained in the adjusted model (mean  SE: females: 49.03  7.14%; males: 41.19  4.89%; F1,9 ¼ 6.263, P ¼ 0.037), attractiveness was not significantly affected by sex when this effect was considered alone in the model (effect of sex: F1,9 ¼ 2.950, P ¼ 0.120). Reaction time was not significantly affected by host infection status (mean  SE: infected: 646.80  20.39 s; uninfected: 644.03  32.47 s; F1,9 ¼ 0.006, P ¼ 0.942) and the effect of host infection status on mosquito reaction time remained nonsignificant after adjusting the model either for host sex (effect of sex: F1,9 ¼ 2.486, P ¼ 0.153; infection status: F1,8 ¼ 0.362, P ¼ 0.564) or for mean body mass (effect of mean body mass: F1,9 ¼ 0.229, P ¼ 0.645; infection status: F1,8 ¼ 0.128, P ¼ 0.729). Activation was not significantly affected by the host pairing type (mean  SE: UU pairing: 52.70  7.31%; UI pairing: 41.63  4.32%; F1,17 ¼ 2.271, P ¼ 0.150). The effect of host pairing on activation remained nonsignificant after adjusting the model for the sum of the body mass of the paired individuals (sum of the body mass: F1,17 ¼ 0.659, P ¼ 0.429; pairing type: F1,16 ¼ 1.561, P ¼ 0.230).

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70

Attractiveness (%)

60 50 40 30 20 10 0

Infected

Uninfected

Malaria infection status Figure 1. Mean  SE attractiveness (%) of malaria-infected (three males and three females) and uninfected (two males and three females) great tits to Culex pipiens mosquitoes, over all the uninfected versus infected dual host choice experiments (N ¼ 28).

DISCUSSION We found that Plasmodium infection altered the attractiveness of wild great tit hosts to naïve, unparasitized C. pipiens mosquitoes, based on a malaria model system in which hosts, parasites and vectors can encounter each other in the wild (Glaizot et al. 2012). Results from the dual host choice experiment showed that uninfected bird hosts (P. major) attracted more host-seeking C. pipiens mosquitoes than the Plasmodium-infected ones. Sex and body mass of the birds did not affect the observed pattern, although females were marginally more attractive than males. Lastly, no significant effects were found for either the bird infection status on the reaction time or for bird pairing type on mosquito activation. Examples illustrating the diversity of behavioural strategies developed by hosts to reduce contact with their parasites abound in the literature (reviewed in Combes 2001; Moore 2002). Avoidance of parasitism has also been documented in diverse plant disease vectors, such as bees transmitting the anther smut disease, Microbotryum violaceum, to white campions, Silene alba (Shykoff & Bucheli 1995; Altizer et al. 1998), grapevine moths, Lobesia botrana, transmitting fungi (Tasin et al. 2012) or leafhoppers and aphids that transmit viruses to plants while xylem sap feeding (Power 1996; Marucci et al. 2005; Daugherty et al. 2011). The observed pattern of bird attractiveness to mosquitoes found in the present study is consistent with previous results, which focused on other avian malaria model systems in both natural and laboratory conditions. For example, Freier & Friedman (1976) found that Aedes aegypti preferentially fed on malaria-free domestic chickens, Gallus gallus domesticus, over individuals artificially infected with Plasmodium gallinaceum. More recently, two fieldexperimental studies showed that the biting midges Culicoides species transmitting Haemoproteus spp., a malaria-like parasite, were more abundant in blue tit, Cyanistes caeruleus, nests when birds were cleared from malaria, which can be interpreted as indirect evidence for healthy host preference (Tomás et al. 2008; Martinez-de la Puente et al. 2009). In contrast, haematophagous insects transmitting malaria or other blood diseases to mammals either did not show any host preference or showed a preference for infected hosts (Day et al. 1983; Ferguson et al. 2003; Ferguson & Read 2004; Lacroix et al. 2005; Lefèvre & Thomas 2008; but see Burkot et al. 1989). Day et al. (1983) and Day & Edman (1983)

suggested that rodent malaria vectors preferred to feed on parasitized hosts to decrease the costs associated with defensive behaviour if infected hosts displayed reduced grooming activities. In this case, parasites and vectors would both benefit from mosquitoes feeding on infected hosts. Mosquitoes may also circumvent host defensive behaviour by synchronizing their hostseeking activity with the inactivity periods of their hosts (Day & Edman 1984). Divergent patterns of host selection between avian and mammalian malaria vectors could be explained by the differential effect of Plasmodium infections on their hosts. During acute infection, malaria-infected humans often exhibit fever peaks and intense perspiration among several other symptoms, while Plasmodium-induced changes in body temperature of birds are still unclear (Hayworth et al. 1987; Williams 2005; Palinauskas et al. 2008). The divergent patterns may also be the result of different coevolutionary histories of host, vector and parasite species, which therefore influence the strength of selection on mosquito choosiness. The propensity of haematophagous insects to search for and to orient towards a host relies on a variety of senses including vision, hearing, mechanoreception and chemoreception (Bowen 1991; Takken 1991; Lehane 2005). Vectors might thus perceive differences in bird metabolic rate (including thermoregulation and CO2 emission), blood quality, defensive behaviour and body odour profiles, which in turn may be affected by parasitism. In mosquitoes, especially the nocturnal species, olfaction is important during the full host-seeking process and for orientation over long and short distances (Gibson & Torr 1999). In the olfactometer, mosquitoes had neither direct nor visual contact with the birds and could only rely on olfactory cues and/or differences in CO2 concentration to fly towards their preferred host. The current experimental design did not allow mosquitoes to detect potential temperature differences that may occur between infected and uninfected birds. Thus, the propensity of vectors to orient preferentially towards healthy hosts is probably due to Plasmodiuminduced changes in bird body odour profiles and/or exhaled CO2, as suggested for humans affected by malaria parasites (Braks et al. 1999; Lefèvre et al. 2006; Prugnolle et al. 2009). Diverse semiochemicals (e.g. Bernier et al. 2008; Syed & Leal 2009) or odours emanating from bird feathers (Allan et al. 2006), faeces (Cooperband et al. 2008) and uropygial gland secretions (Russell & Hunter 2005) have previously been shown to play a role in mosquito attraction, which may in turn be affected by Plasmodium infection. The diversity in the major histocompatibility complex (MHC) alleles also influences both the birds’ susceptibility to Plasmodium spp. infection (Westerdahl 2007; Loiseau et al. 2011) and their body odour profiles (Leclaire et al. 2012). Malaria-infected birds may also have a reduced metabolic rate during acute infection (Hayworth et al. 1987) and, consequently, a lower CO2 production than uninfected birds. As CO2 is an important attractant for numerous mosquito species feeding on humans and other mammals (Gillies 1980), one could expect a biased vector orientation towards healthy birds. It is, however, unclear whether CO2 modulates the host-seeking behaviour of ornithophilic mosquitoes (Allan et al. 2006; Jansen et al. 2009), although some species have numerous sensilla basiconica (McIver 1969) which play a role in the detection of the CO2 molecule. One can thus expect a complex relationship between the birds’ infection status, their body odour profiles, their CO2 production and the host selection behaviour of the mosquitoes. Selection of healthy hosts by vectors can be seen as a by-product of Plasmodium-induced changes in host attraction cues or as an adaptive parasite avoidance behaviour, which may be favoured by natural selection if parasitism entails fitness costs (Hart 1990; Lozano 1991; Hart 1994; Moore 2002). In this case, one could

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expect that vectors have evolved the ability to avoid infected hosts to circumvent costs imposed by parasitism. The general negative impact of malaria parasites on their vectors’ reproductive success has been identified in both laboratory (Hacker 1971; Hacker & Kilama 1974; Freier & Friedman 1976; Hogg & Hurd 1995a, b; Araujo et al. 2011) and field studies (Hogg & Hurd 1997). Plasmodium induces follicle resorption in the ovaries (Carwardine & Hurd 1997), apoptosis of follicular epithelial cells (Hopwood et al. 2001) and impairs the uptake process of vitellogenin (Jahan & Hurd 1998; Ahmed et al. 2001), a key hormone involved in egg production. Moreover, while blood meal size determines the number of eggs laid by female mosquitoes (Briegel 1980), vectors suffer an impaired blood intake when carrying the sporozoite life stage of the parasite in their salivary glands (Rossignol et al. 1984; for a review on fecundity reduction see Hurd 2003). On the other hand, the effect of malaria parasites on the vector’s survival still remains debatable (Ferguson & Read 2002). A reduced life span has been reported in C. pipiens infected by avian malaria (Maier 1973) and in other dipteran insect vectors infected by avian malaria-like para nas & Iezhova 2004). Malarial sites (Desser & Yang 1973; Valkiu parasites may also cause tissue damage and physiological disruptions (Ferguson & Read 2002), induce immunity costs (Yan et al. 1997; Michel & Kafatos 2005) or cause behavioural modifications such as alteration of flight ability (Schiefer et al. 1977; Rowland & Boersma 1988), which may in turn reduce fitness. The parasite avoidance behaviour of C. pipiens reported in this study may affect the transmission rate of the parasite and apparently contrasts with previous findings of a high prevalence of Plasmodium infections in both adult breeding great tits (more than 90%) and C. pipiens mosquitoes (7%) in the study populations (Christe et al. 2012; Glaizot et al. 2012). However, parasite avoidance by vectors may in some cases enhance the parasite’s spread if, for example, healthy hosts are rare and parasitized vectors are selective as well. This has been shown, for example, with epidemiological models of vector-transmitted viruses of plants (McElhany et al. 1995; Sisterson 2008). In the present study, only healthy, naïve mosquitoes were used and the selective behaviour of parasitized mosquitoes remains to be investigated. The advantage of being selective for the mosquito may be difficult to interpret when healthy hosts are scarce, as in our study population. However, seasonal changes in the healthy host frequency, as shown in some studies (Manwell 1955; Janovy 1966; Cosgrove et al. 2008) and the availability of uninfected juveniles in spring may still be a source of selection pressure on the avoidance behaviour of the vector. This might also be the reason for seasonal variations in infection rates in vectors, as observed in some mosquito populations (Reeves et al. 1954; F. Lalubin, A. Delédevant, P. Christe & O. Glaizot, unpublished data for the present study site). In the present study, female birds were marginally more attractive to mosquitoes than males. This may be a source for female-biased parasitism, which has been documented in various parasitic associations (Christe et al. 2007; reviewed in Duneau & Ebert 2012). However, the role of host sex in host selection by avian malaria vectors remains to be investigated. Indeed, male great tits had overall higher malarial parasitaemia than females (McCurdy et al. 1998; Christe et al. 2012), whereas female blue tits were more likely to be infected with Plasmodium relictum than males (Knowles et al. 2011). In our experimental set-up, reaction time and mosquito activation were not affected by host infection status or body mass of the hosts. The average activation (45%) was in the range of what has generally been observed in similar experiments with a diversity of mosquito species (Pates et al. 2005; Cooperband et al. 2008; Lefèvre et al. 2009; Simpson et al. 2009). Other factors, such as temperature and humidity, may affect the activation of mosquitoes (Rudolfs

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1925; Dow & Gerrish 1970; Takken et al. 1997; Gray et al. 2011) and our experimental set-up may only partially reflect what mosquitoes encounter in nature. Conclusion This study provides some insights towards a better understanding of the host selection pattern of avian malaria vectors. The next step should be to investigate host choice selection in wild conditions in which vectors are confronted with a community of different host species that differ in spatial and temporal availabilities as well as in physiological and ecological traits. Other factors such as the parasitized status of the mosquitoes or their degree of hunger may also have major impacts on the vector’s host selection pattern and therefore on the epidemiology of avian malaria. Further studies should now investigate the influence of these parameters on the host selection behaviour of the vectors by exploring, when possible, the natural diversity of relationships that exist between haemosporidian parasites, vertebrate hosts and vectors (Tripet 2009; Megali et al. 2011). Acknowledgments This research is supported by grants from the Swiss National Science Foundation No31003A_120479 and No31003A_138187. We are very grateful to Luca Fumagalli for guidance in molecular work, Pierre Fontanillas, Nicolas Salamin and Jérôme Goudet for helpful advice with statistics, Laura Galbiati and Léo Gaillard for field assistance and two anonymous referees for comments on the manuscript. References Adedolapo, A. A. & Olajumoke, A. M. 2008. A review of manipulations in Plasmodiumemosquito interactions. Pakistan Journal of Medical Sciences, 24, 898e901. Ahmed, A. M., Maingon, R., Romans, P. & Hurd, H. 2001. Effects of malaria infection on vitellogenesis in Anopheles gambiae during two gonotrophic cycles. Insect Molecular Biology, 10, 347e356. Allan, S. A., Bernier, U. R. & Kline, D. L. 2006. Laboratory evaluation of avian odors for mosquito (Diptera: Culicidae) attraction. Journal of Medical Entomology, 43, 225e231. Altizer, S. M., Thrall, P. H. & Antonovics, J. 1998. Vector behavior and the transmission of anther-smut infection in Silene alba. American Midland Naturalist, 139, 147e163. Arathi, H. S., Ho, G. & Spivak, M. 2006. Inefficient task partitioning among nonhygienic honeybees, Apis mellifera L., and implications for disease transmission. Animal Behaviour, 72, 431e438. Araujo, R. V., Maciel, C., Hartfelder, K. & Capurro, M. L. 2011. Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development. Journal of Insect Physiology, 57, 265e273. Beier, J. C. 1998. Malaria parasite development in mosquitoes. Annual Review of Entomology, 43, 519e543. Bensch, S., Stjernman, M., Hasselquist, D., Ostman, O., Hansson, B., Westerdahl, H. & Pinheiro, R. T. 2000. Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proceedings of the Royal Society B, 267, 1583e1589. Bensch, S., Peréz-Tris, J., Waldenström, J. & Hellgren, O. 2004. Linkage between nuclear and mitochondrial DNA sequences in avian malaria parasites: multiple cases of cryptic speciation? Evolution, 58, 1617e1621. Bernier, U. R., Allan, S. A., Quinn, B. P., Kline, D. L., Barnard, D. R. & Clark, G. G. 2008. Volatile compounds from the integument of white leghorn chickens (Gallus gallus domesticus L.): candidate attractants of ornithophilic mosquito species. Journal of Separation Science, 31, 1092e1099. Besansky, N. J., Hill, C. A. & Costantini, C. 2004. No accounting for taste: host preference in malaria vectors. Trends in Parasitology, 20, 249e251. Bowen, M. F. 1991. The sensory physiology of host-seeking behavior in mosquitoes. Annual Review of Entomology, 36, 139e158. Braks, M. A. H., Anderson, R. A. & Knols, B. G. J. 1999. Infochemicals in mosquito host selection: human skin microflora and Plasmodium parasites. Parasitology Today, 15, 409e413. Briegel, H. 1980. Determination of uric acid and hematin in a single sample of excreta from blood-fed insects. Experientia, 36, 1428. Burkot, T. R., Narara, A., Paru, R., Graves, P. M. & Garner, P. 1989. Human host selection by anophelines: no evidence for preferential selection of malaria or

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