A field test of the dilution effect hypothesis in four avian multi-host pathogens.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.

Two avian Plasmodium species trigger different transcriptional responses on their vector Culex pipiens.

Malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium that affects both humans and wildlife. The fitness consequences of infections by avian malaria are well known in birds, however, little information exists on its impact on mosquitoes. Here we study how Culex pipiens mosquitoes transcriptionally respond to infection by two different Plasmodium species, P. relictum and P. cathemerium, differing in their virulence (mortality rate) and transmissibility (parasite presence in exposed mosquitoes' saliva). We studied the mosquito response to the infection at three critical stages of parasite development: the formation of ookinetes at 24 h post-infection (hpi), the release of sporozoites into the hemocoel at 10 days post-infection (dpi), and the storage of sporozoites in the salivary glands at 21 dpi. For each time point, we characterized the gene expression of mosquitoes infected with each P. relictum and P. cathemerium and mosquitoes fed on an uninfected bird and, subsequently, compared their transcriptomic responses. Differential gene expression analysis showed that most transcriptomic changes occurred during the early infection stage (24 hpi), especially when comparing P. relictum and P. cathemerium-infected mosquitoes. Differentially expressed genes in mosquitoes infected with each species were related mainly to the metabolism of the immune response, trypsin, and other serine-proteases. We conclude that these differences in response may partly play a role in the differential virulence and transmissibility previously observed between P. relictum and P. cathemerium in Cx. pipiens.

Habitat-dependent Culicoides species composition and abundance in blue tit (Cyanistes caeruleus) nests.

Wild birds are hosts of Culicoides from as early on as the nesting stage when constrained to their nests. However, the environmental factors which determine the abundance and composition of Culicoides species within each bird nest are still understudied. We sampled Culicoides from Eurasian blue tit (Cyanistes caeruleus) nests found in 2 types of forests located in southern Spain. Firstly, we monitored the abundance of Culicoides species in bird nests from a dry Pyrenean oak deciduous forest and a humid mixed forest comprising Pyrenean and Holm oaks throughout 2 consecutive years. During the 3rd year, we performed a cross-fostering experiment between synchronous nests to differentiate the role of rearing environment conditions from that of the genetically determined or maternally transmitted cues released by nestlings from each forest. We found 147 female Culicoides from 5 different species in the birds' nests. The abundance of Culicoides was higher in the dry forest than in the humid forest. Culicoides abundance, species richness and prevalence were greater when the nestlings were hatched later in the season. The same pattern was observed in the cross-fostering experiment, but we did not find evidence that nestling's features determined by the forest of origin had any effect on the Culicoides collected. These results support the notion that habitat type has a strong influence on the Culicoides affecting birds in their nests, while some life history traits of birds, such as the timing of reproduction, also influence Culicoides abundance and species composition.

Urbanization effects on temporal variations of avian haemosporidian infections.

Urbanization is an important human-driven process that leads to biodiversity loss and alters the interactions between organisms, including disease transmission. Although urbanization affects both host and vector communities, the effects on vector-borne pathogens are still poorly understood. Here, we monitored variation in prevalence and richness of three common blood parasites in birds (Plasmodium, Haemoproteus and Leucocytozoon) from localities with different land uses (urban, rural and natural) during two consecutive years (2013 and 2014). Overall, 1400 juvenile house sparrows (Passer domesticus) from 15 localities in southern Spain were included in this study. Haemoproteus and Leucocytozoon prevalence was higher in 2013 than in 2014, particularly in urban and natural habitats for the case of Leucocytozoon. Prevalence was correlated between years for Haemoproteus and Leucocytozoon, independently of the habitat. Additionally, rural habitats harboured significantly higher Haemoproteus lineage richness compared to urban and natural habitats during 2014. Leucocytozoon lineage richness was negatively correlated between years in rural habitats but positively correlated in urban and natural habitats in comparison. Parasite lineages found in birds were homogeneously distributed along habitats and years and the common lineages prevalence were not influenced by them. Our results highlight different patterns of infection depending on the parasite genera probably related to the composition and density of vector communities. The specific reproductive environmental requirements of the different groups of vectors involved in the transmission may be affected by climatic conditions and landscape features.

Implications of diet on mosquito life history traits and pathogen transmission.

The environment, directly and indirectly, affects many mosquito traits in both the larval and adult stages. The availability of food resources is one of the key factors influencing these traits, although its role in mosquito fitness and pathogen transmission remains unclear. Larvae nutritional status determines their survivorship and growth, having also an impact on adult characteristics like longevity, body size, flight capacity or vector competence. During the adult stage, mosquito diet affects their survival rate, fecundity and host-seeking behaviour. It also affects mosquito susceptibility to infection, which may determine the vectorial capacity of mosquito populations. The aim of this review is to critically revise the current knowledge on the effects that both larval and adult quantity and quality of the diet have on mosquito life history traits, identifying the critical knowledge gaps and proposing future research lines. The quantity and quality of food available through their lifetime greatly determine adult body size, longevity or biting frequency, therefore affecting their competence for pathogen transmission. In addition, natural sugar sources for adult mosquitoes, i.e., specific plants providing high metabolic energy, might affect their host-seeking and vertebrate biting behaviour. However, most of the studies are carried out under laboratory conditions, highlighting the need for studies of feeding behaviour of mosquitoes under field conditions. This kind of studies will increase our knowledge of the impact of diets on pathogen transmission, helping to develop successful control plans for vector-borne diseases.

Plasmodium transmission differs between mosquito species and parasite lineages.

Factors such as the particular combination of parasite-mosquito species, their co-evolutionary history and the host's parasite load greatly affect parasite transmission. However, the importance of these factors in the epidemiology of mosquito-borne parasites, such as avian malaria parasites, is largely unknown. Here, we assessed the competence of two mosquito species [Culex pipiens and Aedes (Ochlerotatus) caspius], for the transmission of four avian Plasmodium lineages (Plasmodium relictum SGS1 and GRW11 and Plasmodium cathemerium-related lineages COLL1 and PADOM01) naturally infecting wild house sparrows. We assessed the effects of parasite identity and parasite load on Plasmodium transmission risk through its effects on the transmission rate and mosquito survival. We found that Cx. pipiens was able to transmit the four Plasmodium lineages, while Ae. caspius was unable to transmit any of them. However, Cx. pipiens mosquitoes fed on birds infected by P. relictum showed a lower survival and transmission rate than those fed on birds infected by parasites related to P. cathemerium. Non-significant associations were found with the host-parasite load. Our results confirm the existence of inter- and intra-specific differences in the ability of Plasmodium lineages to develop in mosquito species and their effects on the survival of mosquitoes that result in important differences in the transmission risk of the different avian malaria parasite lineages studied.

Environmental drivers, climate change and emergent diseases transmitted by mosquitoes and their vectors in southern Europe: A systematic review.

Mosquito borne diseases are a group of infections that affect humans. Emerging or reemerging diseases are those that (re)occur in regions, groups or hosts that were previously free from these diseases: dengue virus; chikungunya virus; Zika virus; West Nile fever and malaria. In Europe, these infections are mostly imported; however, due to the presence of competent mosquitoes and the number of trips both to and from endemic areas, these pathogens are potentially emergent or re-emergent. Present and future climatic conditions, as well as meteorological, environmental and demographic aspects are risk factors for the distribution of different vectors and/or diseases. This review aimed to identify and analyze the existing literature on the transmission of mosquito borne diseases and those factors potentially affecting their transmission risk of them in six southern European countries with similar environmental conditions: Croatia, France, Greece, Italy, Portugal and Spain. In addition, we would identify those factors potentially affecting the (re)introduction or spread of mosquito vectors. This task has been undertaken with a focus on the environmental and climatic factors, including the effects of climate change. We undertook a systematic review of the vectors, diseases and their associations with climactic and environmental factors in European countries of the Mediterranean region. We followed the PRISMA guidelines and used explicit and systematic methods to identify, select and critically evaluate the studies which were relevant to the topic. We identified 1302 articles in the first search of the databases. Of those, 160 were selected for full-text review. The final data set included 61 articles published between 2000 and 2017.39.3% of the papers were related with dengue, chikungunya and Zika virus or their vectors. Temperature, precipitation and population density were key factors among others. 32.8% studied West Nile virus and its vectors, being temperature, precipitation and NDVI the most frequently used variables. Malaria have been studied in 23% of the articles, with temperature, precipitation and presence of water indexes as the most used variables. The number of publications focused on mosquito borne diseases is increasing in recent years, reflecting the increased interest in that diseases in southern European countries. Climatic and environmental variables are key factors on mosquitoes' distribution and to show the risk of emergence and/or spread of emergent diseases and to study the spatial changes in that distributions.

Vector Competence of Aedes caspius and Ae. albopictus Mosquitoes for Zika Virus, Spain.

We assessed the vector competence of Aedes caspius and Aedes albopictus mosquitoes in Spain for the transmission of Zika virus. Whereas Ae. albopictus mosquitoes were a competent vector, Ae. caspius mosquitoes were unable to transmit Zika virus. We also identified high levels of vertical transmission of Zika virus in Ae. albopictus mosquitoes.

Louse flies of Eleonora's falcons that also feed on their prey are evolutionary dead-end hosts for blood parasites.

Host shifts are widespread among avian haemosporidians, although the success of transmission depends upon parasite-host and parasite-vector compatibility. Insular avifaunas are typically characterized by a low prevalence and diversity of haemosporidians, although the underlying ecological and evolutionary processes remain unclear. We investigated the parasite transmission network in an insular system formed by Eleonora's falcons (the avian host), louse flies that parasitize the falcons (the potential vector), and haemosporidians (the parasites). We found a great diversity of parasites in louse flies (16 Haemoproteus and 6 Plasmodium lineages) that did not match with lineages previously found infecting adult falcons (only one shared lineage). Because Eleonora's falcon feeds on migratory passerines hunted over the ocean, we sampled falcon kills in search of the origin of parasites found in louse flies. Surprisingly, louse flies shared 10 of the 18 different parasite lineages infecting falcon kills. Phylogenetic analyses revealed that all lineages found in louse flies (including five new lineages) corresponded to Haemoproteus and Plasmodium parasites infecting Passeriformes. We found molecular evidence of louse flies feeding on passerines hunted by falcons. The lack of infection in nestlings and the mismatch between the lineages isolated in adult falcons and louse flies suggest that despite louse flies' contact with a diverse array of parasites, no successful transmission to Eleonora's falcon occurs. This could be due to the falcons' resistance to infection, the inability of parasites to develop in these phylogenetically distant species, or the inability of haemosporidian lineages to complete their development in louse flies.

Ecological determinants of avian malaria infections: An integrative analysis at landscape, mosquito and vertebrate community levels.

Vector and host communities, as well as habitat characteristics, may have important but different impacts on the prevalence, richness and evenness of vector-borne parasites. We investigated the relative importance of (1) the mosquito community composition, (2) the vertebrate community composition and (3) landscape characteristics on the prevalence, richness and evenness of avian Plasmodium. We hypothesized that parasite prevalence will be more affected by vector-related parameters, while host parameters should be also important to explain Plasmodium richness and evenness. We sampled 2,588 wild house sparrows (Passer domesticus) and 340,829 mosquitoes, and we performed vertebrate censuses at 45 localities in the Southwest of Spain. These localities included urban, rural and natural landscapes that were characterized by several habitat variables. Twelve Plasmodium lineages were identified in house sparrows corresponding to three major clades. Variation partitioning showed that landscape characteristics explained the highest fraction of variation in all response variables (21.0%-44.8%). Plasmodium prevalence was in addition explained by vector-related variables (5.4%) and its interaction with landscape (10.2%). Parasite richness and evenness were mostly explained by vertebrate community-related variables. The structuring role of landscape characteristics in vector and host communities was a key factor in determining parasite prevalence, richness and evenness, although the role of each factor differed according to the parasite parameters studied. These results show that the biotic and abiotic contexts are important to explain the transmission dynamics of mosquito-borne pathogens in the wild.

Two cases of subcutaneous dirofilariasis in Barcelona, Spain.

In recent years, the number of reported cases of human dirofilariasis in Europe has increased and the circulation of Dirofilaria spp. in mosquitoes in several European countries has been proven. We report here two likely autochthonous cases of subcutaneous human dirofilariasis from Barcelona, Spain, caused by Dirofilaria repens. The potential for an increase in human infection is high given the number of cases published recently and the ability of vectors to spread through the Mediterranean basin.

Absence of haemosporidian parasite infections in the long-lived Cory's shearwater: evidence from molecular analyses and review of the literature.

The apparent scarcity or absence of blood parasites in some avian groups, such as seabirds, has been related to intrinsic and extrinsic factors including host immunological capacity, host-parasite assemblage, and ecological parameters, but also to reduced sensitivity of some methods to detect low parasite prevalence/intensities of infection. Here, we examined the haemosporidian parasite prevalence in a breeding population of Cory's shearwater Calonectris diomedea borealis, a long-distance migrant seabird, nesting in the Macaronesian region, in the Eastern Atlantic. Previous studies on Calonectris diomedea complex were based on small sample sizes providing weak evidence for a lack of infections by haemoparasites. Here, we investigated the presence of both parasite infections in C. d. borealis and larvae of potential mosquito vectors on the area. By employing a PCR-based assay, we extensively examined the prevalence of blood parasites belonging to the genera Plasmodium, Haemoproteus, and Leucocytozoon in 286 individuals from different life stages (i.e., chicks, immatures, sabbatical, and breeding adults), facing their specific energetic trade-offs (immunological functions vs. life history activities). We sampled immatures and adult shearwaters, of different sexes, ages, and migratory origins, from two sub-colonies. None of the sampled individuals were infected by these parasites, supporting the hypothesis that there was no in situ or ex situ transmission of vector-borne parasites in marine habitats irrespective of host's life stage and in spite of the presence of the potential Plasmodium vector Culiseta longiareolata breeding in the area. These results suggest that the lack of transmission of haemosporidian parasites on Selvagem Grande may be related to the lack of suitable dipteran vectors at the study sites, which may result from the geographic isolation of this area.

Extremely low Plasmodium prevalence in wild plovers and coursers from Cape Verde and Madagascar.

BACKGROUND: Relatively little is known about the prevalence of blood parasites in shorebirds, especially those breeding in the tropics. The prevalence of blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon was assessed in blood samples from Kentish plovers and cream-coloured coursers in Cape Verde, and samples of Kittlitz's plovers, Madagascar plovers and white-fronted plovers in Madagascar. RESULTS: Only two of these samples were positive for Plasmodium: a Kittlitz's plover was infected by a generalist lineage of Plasmodium that has already been reported in Europe and Africa, while in a white-fronted plover direct sequencing revealed a previously un-described Plasmodium lineage. CONCLUSION: Potential explanations for the low prevalence of blood parasites include the scarcity of vectors in habitats used by these bird species and their resistance to parasitic infections.

Avian phenotypic traits related to feeding preferences in two Culex mosquitoes.

Host choice by mosquitoes affects the transmission dynamics of vector-borne infectious diseases. Although asymmetries in mosquito attraction to vertebrate species have been reported, the relative importance of host characteristics in mosquito blood-feeding behavior is still poorly studied. Here, we investigate the relationship between avian phenotypic traits-in particular, morphometry, plumage coloration, and nesting and roosting behavior-and the blood-feeding patterns in two common Culex mosquito species on a North American avian community. Forage ratios of the mosquito species were unrelated to the phylogenetic relationships among bird species. Culex pipiens fed preferably on birds with lighter-colored plumage and longer tarsi; furthermore, solitary roosting avian species were both bitten by Cx. pipiens and Cx. restuans more often than expected. These associations may be explained by greater mosquito attraction towards larger birds with a greater color contrast against the background. Although communally roosting birds may release more cues and attract more mosquitoes, individuals may in fact receive fewer bites due to the encounter-dilution effect. Mosquito feeding behavior is a highly complex phenomenon, and our results may improve understanding of the non-random interaction between birds and mosquitoes in natural communities.

Culex pipiens forms and urbanization: effects on blood feeding sources and transmission of avian Plasmodium.

BACKGROUND: The wide spread mosquito Culex pipiens pipiens have two forms molestus and pipiens which frequently hybridize. The two forms have behavioural and physiological differences affecting habitat requirements and host selection, which may affect the transmission dynamic of Cx. p. pipiens-borne diseases. METHODS: During 2013, blood engorged Cx. p. pipiens mosquitoes were captured in urban, rural and natural areas from Southern Spain. In 120 mosquitoes, we identified the blood meal origin at vertebrate species/genus level and the mosquito form. The presence and molecular lineage identity of avian malaria parasites in the head-thorax of each mosquito was also analysed. RESULTS: Mosquitoes of the form pipiens were more frequently found in natural than in urban areas. The proportion of Cx. pipiens form molestus and hybrids of the two forms did not differ between habitat categories. Any significant difference in the proportion of blood meals on birds between forms was found. Birds were the most common feeding source for the two forms and their hybrids. Among mammals, dogs and humans were the most common hosts. Two Plasmodium and one Haemoproteus lineages were found in mosquitoes, with non-significant differences between forms. CONCLUSION: This study supports a differential distribution of Cx. p. pipiens form pipiens between urban and natural areas. Probably due to the similar feeding sources of both mosquito forms and their hybrids here, all of them may frequently interact with avian malaria parasites playing a role in the transmission of Plasmodium.

Genetic colour polymorphism is associated with avian malarial infections.

Individual genetic diversity is predicted to influence host-parasite interactions. Together with the genes directly associated with immune responses, variation in genes regulating vertebrate melanin-based pigmentation may play an important role in these interactions, mainly through the pleiotropic effects that affect colour-specific physiology, behaviour and immunity. Here, we test the hypothesis that the prevalence of avian malarial parasites differs between phenotypes in a raptor species in which the genetic basis of colour polymorphism and its pleiotropic effects over immune functions are known. We found that dark morphs had a higher prevalence of Plasmodium parasites than pale ones but detected no such association for Haemoproteus This pattern may be associated with unequal exposure to vectors or, as suggested by our circumstantial evidence, to a differential ability to mount an immune response against blood parasites.

Avian malaria parasites in the last supper: identifying encounters between parasites and the invasive Asian mosquito tiger and native mosquito species in Italy.

BACKGROUND: The invasive Asian tiger mosquito Aedes albopictus has dramatically expanded its distribution range, being catalogued as one of the world's 100 worst invasive alien species. As vectors of pathogens, Ae. albopictus may create novel epidemiological scenarios in the invaded areas. METHODS: Here, the frequency of encounters of Ae. albopictus with the avian malaria parasite Plasmodium and the related Haemoproteus was studied in an area with established populations in northeastern Italy and compared with those from four native mosquito species, Anopheles maculipennis s.l., Culex hortensis, Culex pipiens, and Ochlerotatus caspius. The abdomens of mosquitoes with a recent blood meal were used to identify both the blood meal source and the parasites harboured. RESULTS: Aedes albopictus had a clear antropophilic behaviour while An. maculipennis and Oc. caspius fed mainly on non-human mammals. Birds were the most common hosts of Cx. pipiens and reptiles of Cx. hortensis. Parasites were isolated from three mosquito species, with Cx. pipiens (30%) showing the highest parasite prevalence followed by Cx. hortensis (9%) and Ae. albopictus (5%). CONCLUSIONS: These results are the first identifying the avian malaria parasites harboured by mosquitoes in Italy and represent the first evidence supporting that, although Ae. albopictus could be involved in the transmission of avian malaria parasites, the risk of avian malaria parasite spread by this invasive mosquito in Europe would be minimal.

Individual identification of endangered species using mosquito blood meals: a proof-of-concept study in Iberian lynx.

Host identification from mosquito blood meals has been routinely used to identify the feeding preferences of insects in studies on transmission of vector-borne pathogens. Here, we identified for the first time the susceptibility of the endangered Iberian lynx (Lynx pardinus) to the attack of a wild mosquito female, the mosquito Anopheles atroparvus. Furthermore, we used 11 microsatellite markers to test for the utility of vertebrate DNA isolated from insect blood meals for individual identification of wildlife. Only the three smallest markers were successfully amplified; however, this genotype did not match with any of the previously genotyped individuals in southern Spain. These results support the use of DNA from mosquito blood meals as a non-invasive source of DNA and a powerful tool on epidemiological and conservation biology studies. However, as may be the case of other non-invasive sampling methods, the utility of this technique is probably limited by the quantity and quality of vertebrate DNA.

Survival effects of antibiotic exposure during the larval and adult stages in the West Nile virus vector Culex pipiens.

The ability of mosquitoes to transmit a pathogen is affected, among other factors, by their survival rate, which is partly modulated by their microbiota. Mosquito microbiota is acquired during the larval phase and modified during their development and adult feeding behavior, being highly dependent on environmental factors. Pharmaceutical residues including antibiotics are widespread pollutants potentially being present in mosquito breeding waters likely affecting their microbiota. Here, we used Culex pipiens mosquitoes to assess the impact of antibiotic exposure during the larval and adult stages on the survival rate of adult mosquitoes. Wild-collected larvae were randomly assigned to two treatments: larvae maintained in water supplemented with antibiotics and control larvae. Emerged adults were subsequently assigned to each of two treatments, fed with sugar solution with antibiotics and fed only with sugar solution (controls). Larval exposure to antibiotics significantly increased the survival rate of adult females that received a control diet. In addition, the effect of adult exposure to antibiotics on the survival rate of both male and female mosquitoes depended on the number of days that larvae fed ad libitum in the laboratory before emergence. In particular, shorter larval ad libitum feeding periods reduced the survival rate of antibiotic-treated adult mosquitoes compared with those that emerged after a longer larval feeding period. These differences were not found in control adult mosquitoes. Our results extend the current understanding of the impact of antibiotic exposure of mosquitoes on a key component of vectorial capacity, that is the vector survival rate.