Introduction

Transmission of avian malaria is highly dependent on the distribution, competence and vectorial capacity of vectors. Despite a recent increase in interest toward dipteran insects as major actors in avian malaria systems [1]–[4], there is a lack of general knowledge of the role of the vectors in this mosquito-borne disease. Most of the information on the vectorial competence of diverse mosquito species has been reviewed by Huff [5] but natural avian malaria vectors have been mostly ignored until very recently [6]–[8]. Although many species seem to be competent according to laboratory experiments [6], only a few recent studies have identified natural vectors responsible for avian malaria transmission in different parts of the world, with the notable exception of Europe [1], [2], [9]–[16].

Molecular studies on avian malaria have permitted the investigation of questions concerning phylogeny, phylogeography and life-history evolution of Plasmodium parasites [17]–[21]. However, the epidemiological success of Plasmodium strongly depends on its life cycle within the mosquito and in the vector's ability to find suitable hosts for taking a blood meal. Furthermore, asexual reproduction of Plasmodium occurs in vertebrate hosts whereas sexual reproduction takes place in the mosquito, making the vector the definitive host [22]. Mosquitoes are thus expected to play a major role in both avian malaria parasites' evolution and disease transmission [23], [24]. It is therefore of prime interest to deepen the understanding of the interactions between the different actors of this complex host-parasite system under wild conditions, the first step being the identification of the vector in such wild populations.

The aims of the present study were to identify the natural vector of avian malaria in two wild populations of great tits (Parus major) in Switzerland, where the prevalence of infection has been shown to be high [20], and to identify and compare malaria lineage diversity among the bird hosts and the mosquito vectors by means of mitochondrial cytochrome b gene sequencing.

Materials and Methods

Results

Six genera of mosquitoes representing a total of 15 species were captured from the study populations. Among them, nine species (four genera) are mainly or potentially ornithophilous and were analyzed for the presence of Plasmodium and Haemoproteus parasites (Table 1). Only Culex pipiens individuals were found to be positive for avian malaria with an overall prevalence of 6.6% (n = 394).

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Table 1. Analyses of ornithophilous mosquito species per study location.

https://doi.org/10.1371/journal.pone.0034964.t001

All infected C. pipiens originated from the Dorigny forest (26 out of 330 individuals collected, 7.9%), whereas none of the 64 collected in the Monods forest were found to be infected. Proportions of infected birds at both locations were comparable, with 91% of infected birds in the Monods forest and 98% in Dorigny.

Six different Plasmodium lineages were identified among the 26 infected Culex pipiens, and four among the 54 great tits analysed (Table 2). One lineage of Haemoproteus, known to be transmitted by non-mosquito vectors, was found in a great tit individual.

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Table 2. Plasmodium and Haemoproteus lineages detected by sequencing a portion of the mtDNA cyt b gene (433–478 bp) in 54 Parus major and 26 Culex pipiens individuals.

https://doi.org/10.1371/journal.pone.0034964.t002

Discussion

The relatively high prevalence of Plasmodium spp. found in females Culex pipiens strongly suggests that this species acts as a vector of avian malaria in one of our study populations. C. pipiens was infected with six Plasmodium lineages, among which three were also identified in great tits. Haemoproteus spp., transmitted by biting midges of the genus Culicoides [31], was present in one great tit individual.

Many species of Culex have been described as vectors of avian malaria parasites and their role as competent vectors in experimental infections has been proven [32]. C. pipiens, or the two subspecies C. p. pipiens and C. p. pallens, have been recently reported to be the natural vector of avian malaria in Japan [1], [10]–[14] and North America [2]. Other species of the genus Culex have been described as vectors of avian Plasmodium: C. restuans in North America [2], [16], C. (Melanoconion) ocossa in Panama [8], C. saltanensis in Brazil [33], C. sasai in Japan [14], C. quinquefasciatus in Japan [12] Hawai'i [34] and Mexico [15], C. neavei, C. perfidiosus, C. poicilipes and C. guiarti in Cameroon [35] and finally C. sitiens and C. annulirostris in the southwest Pacific islands [9]. The results of the present study suggest therefore for the first time the role of Culex pipiens in the transmission of avian malaria in the European continent.

None of the mosquitoes captured in Monods were positive for malaria, although prevalence in great tits was 91%. This could be due to our low sample size of parous females and/or to other vector species involved in the transmission of Plasmodium present in this particular site. Culiseta morsitans has been shown to be the vector of Plasmodium circumflexum in waterfowl in New Brunswick [36], whereas Kimura et al. [2] did not find any Plasmodium in 176 Culiseta melanura in Ithaca (USA). Furthermore, C. morsitans was also described as competent vector of P. polare [6], which presents an overall prevalence of 24% and 19.2% among great tits and infected C. pipiens, respectively. The low number of Culiseta morsitans (n = 31) sampled in the present study did not permit the exclusion of this species as potential vector and ongoing studies should elucidate this issue.

Mosquito samplings may not reflect the relative abundances of species in the study sites. First, the type of traps or attractant used may be specific to one or several species [37]. Secondly, capture success of a given species may depend on the trap location itself. A recent study found a higher capture rate for C. pipiens at ground compared to canopy level, whereas biting midges (Culicoides spp.) (Diptera: Ceratopogonidae), known to be involved in the transmission of Haemoproteus, showed the opposite pattern [38]. Similarly, the opportunistic Anopheles plumbeus showed a spatial preference for canopy level [38]. In the present study, gravid mosquito traps were placed at ground level whereas sentinel traps caught mosquitoes approximately 3 meters from the ground. An ongoing study on overall mosquito diversities (Glaizot, unpublished) revealed that C. pipiens was the most caught mosquito species in the Dorigny population (723/953 = 75.9%) whereas it represents 12.1% of the captures in Monods (150/1237). In this latter population, the other main ornithophilic species (Culiseta alaskaensis and C. morsitans) were captured at a rate of 7.1% and 5.2%, respectively. A bias in the trapping method cannot be excluded and further studies should also concentrated on mosquito trapping at the canopy level. Apart strict ornithophilic species listed in Table 1, some mammophilic and opportunistic species are abundant in the study populations. For example, Anopheles plumbeus is widespread in the Dorigny population (136/953 = 14.3%) whereas the mammophilic Ochlerotatus rusticus (579/1237 = 46.8%) and Ochlerotatus annulipes/riparius/cantans (283/1237 = 22.9%) are the most abundant species in Monods.

In the present study, parasite DNA was extracted from dissected mosquito thoraxes. One potential problem to this approach is that sporozoites, the ultimate parasite life stage before transmission to birds, can occur in the haemocoel during their travel from the midgut to the salivary glands but never reach and fully develop in the salivary glands. Consequently, the amplified parasite DNA from mosquito thoraxes may come from non-infectious parasite stages, and therefore non competent mosquitoes [39]. Experimental infections and/or microscopic detection of both oocysts in the midgut and sporozoite in the salivary glands would fully confirm the role of C. pipiens as a natural vector in the study populations. However, the absence of Plasmodium DNA in the other mosquito species captured and the presence of three lineages found in both birds and mosquitoes strongly suggests that C. pipiens is an active actor for the transmission of Plasmodium spp to Parus major. Moreover, an ongoing study (Lalubin et al., unpublished), which combines molecular detection methods with morphological examination of mosquito midguts and salivary glands, confirms the role of C. pipiens in the transmission cycle of avian malaria.

P. relictum (lineages SGS1, GRW11 and P5) was the most abundant haemosporidian parasite found in the great tit populations and one of the main morphospecies responsible for infection in C. pipiens at Dorigny, a result similar to those reported for Japan [1]. In laboratory conditions, P. relictum develops in more than 20 mosquito species including C. pipiens [32] belonging to six genera and has a large bird host range including more than 300 species from different orders and a worldwide distribution [6]. P. polare (SW2) was well represented both in the bird and mosquito samples. According to Valkiunas [6], this parasite has been recorded among 22 bird species in all zoogeographical regions except Antartica and Australia. However, to our knowledge, the present study is the first to report its presence in C. pipiens. Another common lineage, TURDUS 1, assigned to the morphospecies P. circumflexum, has also been found both in great tits and, for the first time, in C. pipiens. This morphospecies has been recorded in over 100 bird species across all regions except the Antartic [6]. Finally, lineage SYAT05, assigned to Plasmodium vaughani, has been found in more than 200 Passeriformes as well as other bird orders [6]. C. pipiens and C. restuans have been reported as vectors for this lineage [2].

Knowledge on temporal and spatial patterns of parasite transmission is essential to understand the selective pressure imposed by parasites on their hosts. For the first time described as a natural vector of avian malaria in Europe, Culex pipiens seems to play a major role in the transmission of a high range of Plasmodium morphospecies. The higher diversity of lineages of Plasmodium found in C. pipiens compared to those found in great tits also suggests that C. pipiens is a natural avian malarial vector for several other bird species. Furthermore, the presence of some of these Plasmodium lineages in bird species sampled in Africa [40] suggests that in these migrating species, infection may also occur in Europe.

Most of the malarial studies to date have used non-natural systems (i.e. hosts and parasites that would not encounter one another in nature). Using vertebrate/mosquito/haemosporidian model systems from natural populations of wild species [41]–[43] may provide powerful insights into the complex interactions and constraints affecting the relationship between hosts and parasites.

Acknowledgments

The late Katia Iritano initiated this study during her Master thesis. We thank Staffan Bensch and Patrick Durand for the appreciated advises for the lab work, Nadia Bruyndonckx and Anne-Lyse Ducrest for laboratory assistance, Laurent Brügger for field work, Geneviève L'Eplattenier for help with mosquito dissections, the two anonymous reviewers and the editor for helpful comments.