Modelling the spatial risk of malaria through probability distribution of Anopheles maculipennis s.l. and imported cases.

Malaria remains one of the most important infectious diseases globally due to its high incidence and mortality rates. The influx of infected cases from endemic to non-endemic malaria regions like Europe has resulted in a public health concern over sporadic local outbreaks. This is facilitated by the continued presence of competent Anopheles vectors in non-endemic countries.We modelled the potential distribution of the main malaria vector across Spain using the ensemble of eight modelling techniques based on environmental parameters and the Anopheles maculipennis s.l. presence/absence data collected from 2000 to 2020. We then combined this map with the number of imported malaria cases in each municipality to detect the geographic hot spots with a higher risk of local malaria transmission.The malaria vector occurred preferentially in irrigated lands characterized by warm climate conditions and moderate annual precipitation. Some areas surrounding irrigated lands in northern Spain (e.g. Zaragoza, Logroño), mainland areas (e.g. Madrid, Toledo) and in the South (e.g. Huelva), presented a significant likelihood of A. maculipennis s.l. occurrence, with a large overlap with the presence of imported cases of malaria.While the risk of malaria re-emergence in Spain is low, it is not evenly distributed throughout the country. The four recorded local cases of mosquito-borne transmission occurred in areas with a high overlap of imported cases and mosquito presence. Integrating mosquito distribution with human incidence cases provides an effective tool for the quantification of large-scale geographic variation in transmission risk and pinpointing priority areas for targeted surveillance and prevention.

Landscape and mosquito community impact the avian Plasmodium infection in Culex pipiens.

Avian malaria parasites provide an important model for studying host-pathogen interactions, yet understanding their dynamics in vectors under natural conditions is limited. We investigated the effect of vector abundance, species richness and diversity, and habitat characteristics on avian Plasmodium prevalence and lineage richness in Culex pipiens across 45 urban, natural, and rural localities in southern Spain. Analyzing 16,574 mosquitoes grouped in 768 mosquito pools, 32.7% exhibited parasite presence. 13 different Plasmodium lineages were identified, with the lineage SYAT05 being the most commonly found. Parasite prevalence positively correlated with the distance to saltmarshes and rivers, but negatively with the distance to total water source. Parasite lineage diversity was higher in natural than in rural areas and positively correlated with mosquito species richness. These results emphasize the complex dynamics of avian Plasmodium in the wild, with habitat characteristics and vector community driving the parasite transmission by mosquito vectors.

Aedes albopictus in a recently invaded area in Spain: effects of trap type, locality, and season on mosquito captures.

Mosquitoes are primary vectors of pathogens impacting humans, wildlife, and livestock. Among them, the Asian tiger mosquito, Aedes albopictus, stands out as an invasive species with a global distribution, having established populations on every continent except Antarctica. Recent findings incriminate Ae. albopictus in the local transmission of several pathogens causing human diseases, including dengue, chikungunya, and Zika viruses and worm parasites as Dirofilaria. In Spain, the establishment of Ae. albopictus occurred in 2004 and it rapidly expanded, currently reaching southern provinces and creating novel epidemiological scenarios in recently invaded areas. In this study, we conducted captures of Ae. albopictus from May to November 2022 in two provinces, Granada and Malaga, situated near the current edge of the species' expanding range in Spain. The objective was to identify the primary factors influencing their captures in these regions. Mosquitoes were captured using BG-Sentinel traps baited with CO2 and BG-Lure, and miniature CDC-UV traps in five different localities. Our findings underscore the influence of both extrinsic factors, such as locality, and intrinsic factors, including mosquito sex, on the abundance of captured Ae. albopictus. A higher abundance of Ae. albopictus was observed in the Malaga province compared to localities in the Granada province. Furthermore, similar numbers of Ae. albopictus mosquitoes were captured in more urbanized areas of Granada, while the lowest counts were recorded in the less urbanized area. These results were compared to captures of another common species in the area, specifically Culex pipiens. Overall, these results represent the first monitoring of invasive Ae. albopictus in the area and are discussed in the light of the potential importance of the species as a nuisance for humans and vectors of pathogens of public health relevance.

Avian Plasmodium in invasive and native mosquitoes from southern Spain.

BACKGROUND: The emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife. METHODS: The presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus. RESULTS: Avian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions. CONCLUSIONS: Unlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required.

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.

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.

Long-term serological surveillance for West Nile and Usutu virus in horses in south-West Spain.

West Nile virus (WNV) is a re-emerging zoonotic pathogen with increasing incidence in Europe, producing a recent outbreak in 2020 in Spain with 77 human cases and eight fatalities. However, the factors explaining the observed changes in the incidence of WNV in Europe are not completely understood. Longitudinal monitoring of WNV in wild animals across Europe is a useful approach to understand the eco-epidemiology of WNV in the wild and the risk of spillover into humans. However, such studies are very scarce up to now. Here, we analysed the occurrence of WNV and Usutu virus (USUV) antibodies in 2102 samples collected between 2005 and 2020 from a population of feral horses in Doñana National Park. The prevalence of WNV antibodies varied between years, with a mean seroprevalence of 8.1% (range 0%-25%) and seasonally. Climate conditions including mean minimum annual temperatures and mean rainy days per year were positively correlated with WNV seroprevalence, while the annual rainfall was negatively. We also detected the highest incidence of seroconversions in 2020 coinciding with the human outbreak in southern Spain. Usutu virus-specific antibodies were detected in the horse population since 2011. The WNV outbreak in humans was preceded by a long period of increasing circulation of WNV among horses with a very high exposure in the year of the outbreak. These results highlight the utility of One Health approaches to better understand the transmission dynamics of zoonotics pathogens.

Interactions between West Nile Virus and the Microbiota of Culex pipiens Vectors: A Literature Review.

The flavivirus West Nile virus (WNV) naturally circulates between mosquitoes and birds, potentially affecting humans and horses. Different species of mosquitoes play a role as vectors of WNV, with those of the Culex pipiens complex being particularly crucial for its circulation. Different biotic and abiotic factors determine the capacity of mosquitoes for pathogen transmission, with the mosquito gut microbiota being recognized as an important one. Here, we review the published studies on the interactions between the microbiota of the Culex pipiens complex and WNV infections in mosquitoes. Most articles published so far studied the interactions between bacteria of the genus Wolbachia and WNV infections, obtaining variable results regarding the directionality of this relationship. In contrast, only a few studies investigate the role of the whole microbiome or other bacterial taxa in WNV infections. These studies suggest that bacteria of the genera Serratia and Enterobacter may enhance WNV development. Thus, due to the relevance of WNV in human and animal health and the important role of mosquitoes of the Cx. pipiens complex in its transmission, more research is needed to unravel the role of mosquito microbiota and those factors affecting this microbiota on pathogen epidemiology. In this respect, we finally propose future lines of research lines on this topic.

The interplay between vector microbial community and pathogen transmission on the invasive Asian tiger mosquito, Aedes albopictus: current knowledge and future directions.

The invasive Asian tiger mosquito Aedes albopictus is nowadays broadly distributed with established populations in all continents except Antarctica. In the invaded areas, this species represents an important nuisance for humans and, more relevant, it is involved in the local transmission of pathogens relevant under a public health perspective. Aedes albopictus is a competent vector of parasites such as Dirofilaria and viruses including dengue virus, Zika virus, and chikungunya virus, among others. The mosquito microbiota has been identified as one of the major drivers of vector competence, acting upon relevant vector functions as development or immunity. Here, we review the available literature on the interaction between Ae. albopictus microbiota and pathogen transmission and identify the knowledge gaps on the topic. Most studies are strictly focused on the interplay between pathogens and Wolbachia endosymbiont while studies screening whole microbiota are still scarce but increasing in recent years, supported on Next-generation sequencing tools. Most experimental trials use lab-reared mosquitoes or cell lines, exploring the molecular mechanisms of the microbiota-pathogen interaction. Yet, correlational studies on wild populations are underrepresented. Consequently, we still lack sufficient evidence to reveal whether the microbiota of introduced populations of Ae. albopictus differ from those of native populations, or how microbiota is shaped by different environmental and anthropic factors, but especially, how these changes affect the ability of Ae. albopictus to transmit pathogens and favor the occurrence of outbreaks in the colonized areas. Finally, we propose future research directions on this research topic.

Bird community effects on avian malaria infections.

In community assembly processes, interspecific interactions play an important role in shaping community diversity, especially at the local scale. Changes in species richness or abundance can modify local infectious disease dynamics, either reducing or increasing the risk of transmission within the community. This study evaluates the effects of bird community on avian haemosporidians infections in a Neotropical region. Bird samples were collected from areas surrounding three dams, and molecular analysis were performed to identify blood-parasitic haemosporidia infecting the birds. Generalized linear models were used to analyze the relationships between the bird community and the prevalence, number of infections, and richness of avian haemosporidian lineages. Non-significant effects of bird community dominance and richness on the prevalence of avian parasites and the number of infections of Haemoproteus were found. However, there was evidence of an amplification effect. Host dominance was associated with the total number of infections, the number Plasmodium infections and the expected richness of Plasmodium lineages, while the expected richness of Haemoproteus lineages was associated with the richness of bird species. These findings highlight the role of host community dominance and richness in the dynamics of parasite infections, potentially influenced by the availability of competent hosts. This study contributes significantly to our understanding of blood parasite diversity in tropical birds within a relatively understudied region of South America.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Oxidative status in relation to blood parasite infections in house sparrows living along an urbanization gradient.

Living organisms are exposed to a wide range of substances - internal and external - which act like reactive oxygen species (ROS). Oxidative damage accurs when the balance between ROS and antioxidant defenses is altered. Urbanization and parasite infection are both important sources of ROS with different harmful effects on wildlife health, but the potential synergies between both factors are poorly known. Here, we analyse the oxidative stress of wild juvenile male house sparrows (Passer domesticus) along an urbanization gradient in relation to the infection status by three common blood parasites (Plasmodium, Haemoproteus and Leucocytozoon) and bird body condition. We analysed samples from 688 birds captured at 45 localities from southern Spain grouped into triplets including an urban, a rural and a natural habitat, with 15 localities per habitat type. We measured i) thiobarbituric acid reactive substances (TBARS) levels as indicator of the oxidative damage to lipids, and the activity of three antioxidant enzymes ii) glutathione peroxidase (GPx), iii) superoxide dismutase (SOD) and iv) glutathione reductase (GR) as indicators of bird's antioxidant capacity. Birds infected with Haemoproteus and urban birds showed significantly and marginally higher levels of TBARS than uninfected and rural birds, respectively. The relationship between TBARS and body condition is different regarding the infection status (significative) and habitat (marginally significant) being negative for Haemoproteus infected and urban birds but positive for uninfected and non-urban birds. The antioxidant activity was significantly lower in Plasmodium infected birds but marginally higher in Leucocytozoon infected birds than in uninfected ones. Individuals with higher body condition had higher GPx and SOD activity in relation to a lower GR activity. Overall, these results suggest that blood parasites infections and urbanization affect the oxidative status of wild birds and highlight the role of bird's body condition on the regulation of the oxidative stress status.

Methodological procedures explain observed differences in the competence of European populations of Aedes albopictus for the transmission of Zika virus.

The risk of autochthonous transmission of Zika virus (ZIKV) in Europe greatly depends on the capacity of Aedes albopictus to transmit ZIKV. Experimental studies of the vector competence of European populations of this invasive mosquito species provided contrasting results, that may suggest differences between mosquito populations in ZIKV competence and/or differences in the methodological procedures used on the experimental studies. We systematically reviewed published studies of experimental mosquito infections and show that differences detected in the infection and transmission rates of Ae. albopictus are mostly due to differences between studies in the temperature, the viral load used to inoculate mosquitoes, and the number of days post-exposure (dpe) to the virus at which mosquitoes were analysed. In particular, the proportion of infected mosquitoes was affected by the viral load inoculated and the dpe, whereas the transmission rate was influenced by the viral load and temperature. Thus, the methodological differences used in these studies greatly affect the conclusions obtained regarding the role of Ae. albopictus in Europe as a competent vector for ZIKV and its epidemiological relevance.

A One Health view of the West Nile virus outbreak in Andalusia (Spain) in 2020.

Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds (Turdus merula) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

Widespread resistance to anticoagulant rodenticides in Mus musculus domesticus in the city of Barcelona.

Control of rodent populations is a big challenge because of the rapid evolution of resistance to commonly used rodenticides and the collateral negative impacts that these products may have on biodiversity. Second-generation anticoagulants are very efficient but different single nucleotide polymorphisms (SNPs) in the Vkorc1 gene may confer resistance in rodents. We sequenced exons 1, 2 and 3 of the Vkorc1 gene from 111 mice (Mus musculus domesticus) captured across the city of Barcelona and found SNPs associated with resistance to first- and second-generation anticoagulants in all of them. Although most of the SNPs were associated with resistance to bromadiolone, we also found SNPs associated with resistance to brodifacoum. Out of all the individuals analyzed, 94.59 % carried mutations associated to introgression events with Mus spretus, a sympatric rodent species. Currently most of the chemical products for rodent control commercialized in the area are based on bromadiolone, although recent public control campaigns have already shifted to other products. Thus, the widespread occurrence of resistant mice to bromadiolone represents a challenge for rodent control in Barcelona and may increase the risk of secondary poisoning of animals preying on this species. Public health managers, pest control companies and citizens should be aware that the use of bromadiolone based products is ineffective and represents a risk for the environment, including human and animal health.

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.

Ecological Effects on the Dynamics of West Nile Virus and Avian Plasmodium: The Importance of Mosquito Communities and Landscape.

Humans and wildlife are at risk from certain vector-borne diseases such as malaria, dengue, and West Nile and yellow fevers. Factors linked to global change, including habitat alteration, land-use intensification, the spread of alien species, and climate change, are operating on a global scale and affect both the incidence and distribution of many vector-borne diseases. Hence, understanding the drivers that regulate the transmission of pathogens in the wild is of great importance for ecological, evolutionary, health, and economic reasons. In this literature review, we discuss the ecological factors potentially affecting the transmission of two mosquito-borne pathogens circulating naturally between birds and mosquitoes, namely, West Nile virus (WNV) and the avian malaria parasites of the genus Plasmodium. Traditionally, the study of pathogen transmission has focused only on vectors or hosts and the interactions between them, while the role of landscape has largely been ignored. However, from an ecological point of view, it is essential not only to study the interaction between each of these organisms but also to understand the environmental scenarios in which these processes take place. We describe here some of the similarities and differences in the transmission of these two pathogens and how research into both systems may facilitate a greater understanding of the dynamics of vector-borne pathogens in the wild.