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.

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. Graphical

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.

West Nile virus (WNV) is a flavivirus naturally circulating between mosquito vectors and birds, occasionally infecting horses and humans and causing epidemiologically relevant outbreaks. In Spain, the first big WNV outbreak was recorded in 2020, resulting in 77 people infected and 8 fatalities, most of them in southern Spain. Culex perexiguus was identified as the primary vector of WNV maintaining its enzootic circulation of the virus. Growing evidence highlights the role of mosquito microbiota as a key component determining the vectorial capacity of mosquitoes, largely contributing to disease epidemiology. Here, we develop, to our knowledge, the first identification of the microbiota composition of this mosquito vector under natural conditions and test for the potential relationship between mosquito microbiota composition and WNV infection. To do so, we collected mosquitoes in a natural area of southern Spain during the 2020 WNV outbreak and identified the microbiota composition of mosquitoes using a 16S rRNA gene metabarcoding approach. The microbiota of Cx. perexiguus was dominated by the phylum Proteobacteria. The most abundant families were Burkholderiaceae and Erwiniaceae, including the genera Burkholderia, Erwinia, and Pantoea. The genus Wolbachia, which use to dominate the microbiota of Cx. pipiens and negatively interact with WNV according to the literature, had a low prevalence and relative abundance in Cx. perexiguus and its abundance did not differ between WNV-positive and WNV-negative mosquito pools. The microbiota diversity and composition of Cx. perexiguus were not significantly related to the WNV infection status. These results provide the first identification of the mosquito microbiota in an endemic area of WNV circulation in Spain.

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.

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 study the mosquito response to the infection at three critical stages of parasite development: formation of ookinetes at 24 hours post-infection (hpi), the release of sporozoites into the hemocoel at 10 days post-infection (dpi), and storage of sporozoites in the salivary glands at 21dpi. 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 most of the 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 immune response, trypsin, and other serine-proteases metabolism. We conclude that these differences in response may partly play a role in the differential virulence and transmissibility previously observed in P. relictum and P. cathemerium in Cx. pipiens .