https://orcid.org/0000-0002-4664-9011
Figures
Abstract
The common house mosquito Culex pipiens s.l., widely distributed in Europe, Africa, and North America has two recognized biotypes, Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus, which hybridize. Despite their morphological similarities, these biotypes may exhibit ecological differences. This complex ecological mosaic may affect the interaction of Cx. pipiens with pathogens like avian Plasmodium, which is transmitted to wildlife. Although the vector competence for Cx. pipiens biotype molestus has been well studied, there is a lack of studies comparing the vector competence of Cx. pipiens biotype pipiens and their hybrids for the transmission of avian Plasmodium. Here, we evaluated the vector competence of the Cx. pipiens biotypes pipiens, molestus and their hybrids for the transmission of two avian Plasmodium species. Mosquitoes were allowed to feed on blood of wild infected birds and the presence of DNA of Plasmodium in head-thorax and saliva of mosquitoes was molecularly evaluated at 13 day-post exposure. The transmission rates (i.e., the detection of parasite DNA in mosquito saliva) for Plasmodium cathemerium were similar for the two biotypes of Cx. pipiens and their hybrids while Plasmodium relictum DNA was only found in the saliva of Cx. pipiens biotype pipiens. In addition, P. cathemerium was significantly more prevalent than P. relictum in the saliva of Cx. pipiens biotype pipiens. Our results suggest that avian Plasmodium is transmitted by both Cx. pipiens biotypes and their hybrids although differences could be found depending of the parasite species studied. Differences in the abundance of each biotype and their hybrids within areas characterized by distinct environmental conditions, along with variations in their blood-feeding patterns and the parasites infecting birds, could ultimately determine differences in the relevance of each Cx. pipiens biotype in the transmission of avian Plasmodium.
Citation: Gutiérrez-López R, Yan J, Gangoso L, Soriguer R, Figuerola J, Martínez-de la Puente J (2024) Are the Culex pipiens biotypes pipiens, molestus and their hybrids competent vectors of avian Plasmodium? PLoS ONE 19(12): e0314633. https://doi.org/10.1371/journal.pone.0314633
Editor: Angela Monica Ionica, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Life Science Institute, ROMANIA
Received: September 3, 2024; Accepted: November 14, 2024; Published: December 3, 2024
Copyright: © 2024 Gutiérrez-López et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was funded by projects PGC2018-095704-B-100 obtained by JF and PID2020-118205GB-I00 obtained by JMP, from Agencia Estatal de Investigación (Ministerio de Ciencia e Innovación), with support of the European Regional Development Funds (FEDER). RGL is currently supported by postdoctoral grant Sara Borrell from Instituto de Salud Carlos III (REF. CD22CIII-00009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare no competing interests.
Introduction
The common house mosquito Culex pipiens s.l. is widely distributed in temperate regions across the northern hemisphere, including Europe and Africa and, more recently, America, Asia, and Australia [1, 2], where it usually breeds in anthropized habitats. In Europe, the Culex pipiens complex (Cx. pipiens s.l.) consists of several species, including Culex pipiens s.s. (Linnaeus, 1758) and Culex quinquefasciatus (Say, 1823) [3]. In addition, for Cx. pipiens s.s., two biotypes are recognized, namely Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. The Cx. pipiens biotype pipiens is typically associated with aboveground habitats, such as standing water in clogged gutters or artificial containers, and may exhibit a bird-biased blood-feeding pattern [4, 5, but see 6]. In contrast, Cx. pipiens biotype molestus (Forskål, 1775) is frequently found in underground habitats, such as basements, tunnels, and sewers. The Cx. pipiens biotype molestus tends to feed on mammalian blood, including humans as hosts, and exhibits autogeny, that is, the ability to produce eggs without a blood meal [7]. Nevertheless, the classification of Cx. pipiens biotype pipiens as strictly ornithophilic and the Cx. pipiens biotype molestus as mammalophilic may be considered simplistic, as both Cx. pipiens biotypes feed on a variety of birds and mammals [6]. These two Cx. pipiens biotypes are capable of interbreeding, and hybrids are suggested to exhibit intermediate host-feeding behavior, also incorporating both mammals and birds into their diet [2–4, 7]. This complex ecological mosaic may influence the ability of mosquitoes to interact with pathogens that infect different vertebrate groups, potentially affecting their transmission. For instance, the Cx. pipiens biotype pipiens could play an important role in the natural transmission cycle of West Nile virus (WNV) among birds, while Cx. pipiens hybrids have been proposed as more suitable bridge vectors of WNV from birds to humans [7].
Avian Plasmodium is a mosquito-borne haemosporidian parasite with significant impacts on both livestock and wildlife [8]. These parasites are considered as model pathogens for studying the ecology and evolution of vector-pathogen interactions [9], and share some characteristics, including the insect vectors, with other avian pathogens such as WNV [10]. Sporozoites, the infective forms of avian Plasmodium, accumulate in the mosquito’s salivary glands and are transmitted to the bloodstream of new bird hosts through mosquito bites. Several mosquito species are involved in transmitting malarial parasites, with species of the Culex genus playing a key role as vectors of avian Plasmodium spp. [11]. Culex pipiens has been experimentally confirmed as a vector of different Plasmodium species [11–13].
In southern Europe, including Spain, both Cx. pipiens biotype pipiens and molestus and their hybrids are frequently found [2], where they exhibit similar feeding patterns, which promotes frequent interactions with avian Plasmodium under natural conditions [14]. Different studies have experimentally demonstrated the transmission capacity of both Cx. pipiens biotypes for several avian Plasmodium species. For instance, Plasmodium relictum is known to complete sporogony in both Cx. pipiens biotypes [11, 15–17]. Vector competence reflects the intrinsic ability of an arthropod vector to transmit an infectious agent through its bites [18]. Although, most studies have been conducted using Cx. pipiens biotype molestus, probably favored by its ability to breed in colonies, there is a lack of studies comparing the vector competence between Cx. pipiens biotypes and their hybrids for the transmission of avian Plasmodium. Thus, our aim is to experimentally assess the vector competence of Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus as well as their hybrids for the transmission of two avian Plasmodium species, namely P. relictum and Plasmodium cathemerium using the forced salivation technique [19].
Materials and methods
Mosquito collection and rearing conditions
Mosquitoes analysed in Gutiérrez-López et al. [16] were used in this study. Here, we molecularly determined the biotype of the Cx. pipiens mosquitoes to compare the vector competence of the different Cx. pipiens biotypes for avian Plasmodium transmission. Culex pipiens larvae were collected in La Cañada de los Pájaros, Andalusia, Spain (6°14′W, 36°57′N) during summer 2014. Mosquito larvae were transported to the facilities of the Estación Biologica de Doñana (EBD-CSIC) where they were maintained in a climatic chamber at 28°C, 65–70% RH and 12:12 light: dark cycle [19]. Adult female mosquitoes were identified to the species level following Schaffner et al. [20] and placed in insect cages (BugDorm-43030F, 32.5 × 32.5 × 32.5 cm). Mosquitoes were fed ad libitum with 1% sugar solution. Two-three-week-old female mosquitoes were deprived of the sugar solution one day prior to each experimental exposure to birds.
Bird sampling and maintenance
Three juvenile house sparrows (Passer domesticus) were captured using mist nets in the Huelva province. The birds were individually ringed with a numbered metal ring. The birds were blood sampled from the jugular vein. A drop of blood was smeared to quantify their parasitaemia [21] and, subsequently, the intensity of infection by Plasmodium and Haemoproteus parasites was estimated as the percentage of infected cells per 100 erythrocytes after counting 4,000–10,000 erythrocytes at ×1000 magnification. The birds were transported to the Unit of Animal Experimentation at the EBD-CSIC and maintained in birdcages (58.5×25×36 cm) in a vector-free room under controlled conditions (23 ± 1°C, 40–50% RH and 12:12 light: dark cycle). The birds had ad libitum access to a standard mixed diet for seed-eating and insectivorous birds (KIK, GZM S.L., Alicante, Spain). Three days after exposure to mosquitoes, the birds were blood-sampled again (0.2 ml) to detect any potential change in infection status or parasite lineage identity. No changes were observed during the experiment. The birds were released at the site of capture at the end of the experiment.
Exposure procedure
After 11 days of acclimation, each bird was individually housed in a birdcage (38.5×25.5×26 cm) placed within an insect tent (BugDorm-2120, 60×60×60 cm). Over the course of four separate nights, each bird was introduced into independent tents and exposed to unfed Culex pipiens females. The number of mosquitoes used per bird each night varied according to the availability of 2-3-week-old unfed mosquitoes: 50 on the first night, 57 on the second night, 105 on the third night, and 100 on the fourth night (overall, 312 mosquitoes were allowed to feed on each bird). Birds were exposed to mosquitoes from 8:00 pm to 8:00 am. Mosquitoes were only exposed to birds once. After each exposure, mosquitoes with visible blood meals were separated and kept in insect cages (BugDorm-43030F) under the previously mentioned conditions during 13 days. Previous studies found that avian Plasmodium complete its development in mosquitoes in 8–13 days [8, 22]. During this period, engorged mosquitoes had ad libitum access to 1% sugar solution. At 13 days post exposure (dpe), we extracted the saliva of mosquitoes following ref. [19]. Subsequently, mosquitoes were dissected and the head-thorax portion containing the salivary glands, was stored individually in Eppendorf tubes. Due to logistical problems, the head-thoraxes and saliva of seven mosquitoes and the saliva of two additional mosquitoes that survived until 13 dpe were not examined. All samples were kept at −80°C until further molecular analyses. Regional authorities (Junta de Andalucía) and the CSIC Ethics Committee approved the procedures used in this study (ref. CEBA-EBD-12-40).
Molecular identification of blood parasites and mosquito biotypes
We used the MAXWELL® 16 LEV Blood DNA Kit for the extraction of genomic DNA from birds’ blood samples (both the initial and final samples) following [23]. DNA from the head-thorax and saliva of mosquitoes was extracted using the Qiagen DNeasy® Kit Tissue and Blood (Qiagen, Hilden, Germany). A 478-bp fragment of the mitochondrial cytochrome b gene of Plasmodium / Haemoproteus parasites was amplified using the procedure described in [24]. DNA extracted from the head-thorax of mosquitoes analysed for the presence of Plasmodium parasites was used to identify the Cx. pipiens biotype by amplifying the 5′ flacking region of CQ11 microsatellite [25]. Amplicons were verified on 2% agarose gels and sequenced in both directions with the Macrogen sequencing service (Macrogen Inc., Amsterdam, The Netherlands). Parasite sequences were edited using the Sequencher™ software v 4.9 (Gene Codes Corp. © 1991–2009, Ann Arbor, MI 48108). Parasite lineages and morphospecies were identified by blast comparison with sequences deposited in GenBank (National Center for Biotechnology Information) and Malavi [26].
Statistical analysis
The presence of parasite DNA in the head-thorax of mosquitoes was used as an indicator of the infection rate. Infection rate was calculated as the number of Plasmodium positive mosquitoes divided by the number of blood-fed females analysed. The presence of parasite DNA in mosquito saliva was used as an indicator of the transmission rate and was calculated as the number of mosquitoes with Plasmodium positive saliva divided by the number of mosquitoes with Plasmodium positive head-thoraxes. We tested for differences in Plasmodium infection and transmission rates among both Culex biotypes and their hybrids using Generalized Linear Mixed Models GLMMs with binomial error and a logit link function. The Cx. pipiens biotype was included as a factor with three levels (pipiens, molestus and hybrids) while bird identity was included as a random term. In addition, we assessed the effect of Plasmodium species on the infection status by including the parasite identity as the only predictor variable in the former models. This model only was done for Cx. pipiens biotype pipiens due to the absence of P. relictum in the saliva of Cx. pipiens biotype molestus and hybrids (see results). We conducted a third model to test differences in P. cathemerium infection in the head-thorax and saliva of mosquitoes from both Cx. pipiens biotypes and their hybrids. Fixed effects were tested using likelihood ratio tests. Overdispersion was checked using the overdispFun function. Statistical analyses were conducted using the package lme4 [27] in R version 4.3.2 (R Core Development Team, 2016) [28].
Results
Among the three bird donors, one individual (house sparrow 1) was infected with the P. relictum lineage GRW11 (parasite load <0.001%), another bird (house sparrow 2) was infected with the P. cathemerium lineage PADOM01 (parasite load = 0.2%), and the third bird (house sparrow 3) was infected with the P. relictum lineage SGS1 (parasite load = 0.3%). All birds also exhibited coinfection with Haemoproteus PADOM05, a parasite transmitted by Culicoides spp. but not by mosquitoes.
Overall, 126 out of 936 mosquitoes (13.5%) used in this study fed on bird blood, with 105 individuals surviving until 13 days post-exposure (dpe). A total of 98 head-thorax and 96 saliva samples were analysed molecularly. Among these, 52 corresponded to Cx. pipiens biotype pipiens (40 exposed to birds infected with P. relictum and 12 exposed to a bird infected with P. cathemerium), 7 corresponded to Cx. pipiens biotype molestus (5 exposed to P. relictum and 2 to P. cathemerium), and 39 corresponded to hybrids (27 exposed to P. relictum and 12 to P. cathemerium) (Table 1 and Table 2).
Among the mosquitoes analysed, 23 Cx. pipiens biotype pipiens mosquitoes (44.2%; N = 52) tested positive for Plasmodium in the head-thorax. Of these, five individuals (21.7%) also had Plasmodium DNA in their saliva. Three Cx. pipiens biotype molestus mosquitoes (42.9%; N = 7) had Plasmodium DNA in the head-thorax, with only one individual (33.4%) also showing Plasmodium DNA in the saliva. Eighteen hybrids (46.2%; N = 39) were positive for Plasmodium DNA in the head-thorax, with two of them (11.1%) also showing positive results in their saliva.
Culex pipiens biotype pipiens was able to transmit P. relictum and P. cathemerium, as supported by the detection of DNA of both Plasmodium species in their head-thorax and saliva (Table 1). Cx. pipiens biotype molestus tested positive for P. cathemerium in their head-thorax and saliva, but P. relictum DNA was only detected in the head-thorax of mosquitoes (Table 1). In addition, P. relictum DNA was found in the head-thorax of Cx. pipiens hybrids, but it was not detected in the mosquito saliva, meanwhile P. cathemerium DNA was present in both the head-thorax and saliva of hybrids (Table 1). Table 3 summarized the number of mosquitoes analysed exposed to each bird.
The two Cx. pipiens biotypes and their hybrids showed a similar Plasmodium prevalence in the head-thorax (χ2 = 1.25; d.f. = 2; P = 0.53). In addition, non-significant differences were found for the presence of Plasmodium DNA in the saliva of mosquitoes of the two biotypes and their hybrids (χ2 = 1.90; d.f. = 2; P = 0.39). We did not find significant differences in the prevalence of P. cathemerium in the head-thorax (χ2 = 0.90; d.f. = 2; P = 0.64) or in the saliva (χ2 = 1.43; d.f. = 2; P = 0.49) between Cx. pipiens biotypes and their hybrids, suggesting similar vector competence for P. cathemerium. Regarding the Cx. pipiens biotype pipiens, the prevalence of Plasmodium in the head-thorax was similar between both Plasmodium species (Fig 1; χ2 = 1.28; d.f. = 1; P = 0.26). However, significant differences were found for the prevalence of the two parasite species in the saliva of Cx. pipiens biotype pipiens (χ2 = 5.06; d.f. = 1; P = 0.02), being P. cathemerium more prevalent than P. relictum (40.0% and 9.1%, respectively) (Fig 1).
relictum and P. cathemerium. Sample size indicated over each bar. Statistically significant differences are indicated with an asterisk (*). NS means non-significant differences.
Discussion
Previous studies on the competence of Cx. pipiens biotypes pipiens and molestus and their hybrids for transmitting avian pathogens have revealed potential differences between them. For instance, although all these mosquitoes can transmit WNV, differences arose depending on temperature [29, 30]. Here, using avian malaria parasites as study model, we provide evidence for a similar competence of the Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus and their hybrids to transmit avian malaria parasites. However, we found differences between Plasmodium species in their capacity to develop in these mosquitoes.
Previous studies using Cx. pipiens colonies have observed that the widespread P. relictum lineages SGS1 and GRW11, can complete sexual reproduction in Cx. pipiens biotype molestus, with sporozoites, the infective forms, being detected in the salivary glands of female mosquitoes [15, 31]. Similarly, P. relictum lineage GRW4 completed sporogony in Cx. pipiens biotype molestus, with a considerable number of sporozoites being accumulated in the salivary glands [16]. Avian Plasmodium parasites, including P. relictum, have been found in Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus and their hybrids [14, 32, 33]. In mosquitoes from Eastern Austria, the avian Plasmodium prevalence was 2.33% in Cx. pipiens biotype molestus, 1.99% in Cx. pipiens biotype pipiens, and 5.26% in the hybrids [33]. However, in this study, P. relictum was only found in Cx. pipiens biotype pipiens [33]. In mosquitoes from Japan [32], avian Plasmodium was also found in Cx. pipiens biotype molestus with a prevalence of 13.5%. These results support the potential role of Cx. pipiens biotype molestus in the transmission of avian Plasmodium under natural conditions.
Here, by exposing female mosquitoes emerged from field-collected larvae to naturally infected birds, we provide evidence for the transmission of P. cathemerium lineage PADOM01 in Cx. pipiens of the two biotypes, pipiens and molestus, and their hybrids with a similar prevalence between them. These results are especially relevant to confirm the vector competence of Cx. pipiens for avian Plasmodium since most studies used mosquitoes maintained in colonies. Vector competence may differ between wild-collected and colony-maintained mosquitoes, due to the higher likelihood of bottlenecks and genetic drift processes in the colonies [34–36], potentially influencing the conclusions drawn. We found significant differences in the transmission rate between Plasmodium species in Cx. pipiens biotype pipiens, being P. cathemerium more prevalent than P. relictum as previously reported [12], a pattern evident only when the presence of parasite DNA was tested in mosquito saliva.
Different factors could affect the vector competence of avian Plasmodium by the Cx. pipiens biotypes, including the time required for Plasmodium species to develop and reach the salivary glands (i.e., the extrinsic incubation period). Plasmodium cathemerium sporozoites have been identified in the salivary glands of Cx. pipiens at 7 dpe [37], while P. relictum sporozoites have been found as early as 4 and 5 dpe [38, 39]. However, in Cx. pipiens, sporozoites of P. relictum lineages SGS1 and GRW11 were not detected in the salivary glands until 14 dpe (the presence of sporozoites was verified at 12 dpe, but not at 13 dpe) [40].
The intensity of Plasmodium infection in birds could also have an effect on the success of parasite development within the vector and, consequently, in the ability to transmit the parasite. A positive relationship between the density of Plasmodium gametocytes and the proportion of oocysts in mosquitoes was found in humans [41]. However, this was not the case of a previous study using avian Plasmodium [42]. Similarly, the antimalarial treatment and the subsequent reduction of parasite load in birds did not affect the proportion of mosquitoes with Plasmodium-positive head-thoraxes or saliva [43]. These results suggest that avian Plasmodium may still develop in mosquitoes, even when vectors feed on birds with low infection intensities, undetectable by microscopy [41, 43]. Furthermore, mosquito age may also affect the parasite development, as older mosquitoes are more resistant to avian Plasmodium infections compared to younger ones [44]. In this study, 2- to 3-week-old mosquitoes were used, which could potentially reduce the observed infection prevalence. Nevertheless, we did not expect this factor to cause differences between Cx. pipiens biotypes and avian Plasmodium species, as mosquitoes were randomly distributed across the experiments. Further experimental studies are necessary to explore the impact of mosquito age on vector competence for avian Plasmodium.
In conclusion, Cx pipiens biotypes may have differential roles in the epidemiology of avian pathogens such as WNV [45]. Our results support that the competence of both Cx. pipiens biotypes and their hybrids to transmit avian Plasmodium may be similar, although differences may arise depending on the species of Plasmodium studied. Under an ecological perspective, the results reported here should be contextualized with data about the abundance of each Cx. pipiens biotype and their hybrids according to different landscape characteristics and other ecological factors which could affect their abundance in a particular area, including the different species of parasites circulating in the area [2, 14].
Acknowledgments
Martina Ferraguti, Alberto Pastoriza, Esmeralda Pérez and Isabel Martín helped during the field and laboratory work. Plácido and Maribel allowed us to sample mosquito larvae in the Cañada de los Pájaros. We would also like to thank anonymous reviewers for constructively revising this manuscript.
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