[First detection of Rift Valley Fever Virus among Culex pipiens in Tahoua, Niger]. / Détection du virus de la fièvre de la vallée du Rift chez Culex pipiens à Tahoua au Niger.

Background: The Rift Valley Fever (RVF) is an arbovirus disease responsible of regular epizootics and epidemics in sub-Saharan Africa and Arabian Peninsula. In 2016, Niger experienced its first outbreak of RVF in Tahoua region, which resulted in high consequences in animal and human health. The aim of this study was to investigate on the RVFV circulation among potential vectors of the disease. Methods: This was a cross-sectional survey carried out in Tahoua and Agadez regions in August 2021. Adult mosquitoes were collected by using the morning spray in human dwellings and the CDC light trap methods. After morphological identification, viral RNA was extracted. The RNA was extracted by using QIAamp Viral RNA Mini Kit (Qiagen). The RVFV detection was performed by using the qRT-PCR method. Results: A total of 2487 insects (1978 mosquitoes, 509 sandflies and 251 biting midges) were identified and divided into three families (Culicidae, Psychodidae and Ceratopogonidae). The Culicidae family composed of the Culex genus being the most abundant with a predominance of Cx.pipiens (31.88%; n = 793) followed by Mansonia sp (21.51%; n = 535), Anophelesgambiae s.l. (8.44%; n = 210), An. pharoensis (0.72%; n = 18), An. rufipes (0.48%; n = 12), Cx. quinquefasciatus (6.39%; n = 159), the Psychodidae with sandflies (20.46%; n = 509), and the Ceratopogonidae with Culicoides genus (10.09%; n = 251). The qRT-PCR carried out on a sample of mosquitoes (N = 96) highlighted that one individual of Cx.pipiens was found positive to RVFV. This specimen was from Tassara locality (Tahoua) and collected by CDC Light Trap method. Conclusion: This study reveals for the first time the circulation of RVFV among Cx.pipiens in Niger and highlights the possible vectorial role of this vector in the disease transmission. Further investigations should be carried out to identify the biological and ecological determinants that support the maintenance of the virus in this area in order to guide control interventions.

Characterization of mosquito host-biting networks of potential Rift Valley fever virus vectors in north-eastern KwaZulu-Natal province, South Africa.

BACKGROUND: Rift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus with serious implications for livestock health, human health, and the economy in Africa, and is suspected to be endemic in north-eastern KwaZulu-Natal (KZN), South Africa. The vectors of RVFV in this area are poorly known, although several species, such as Aedes (Neomelaniconion) mcintoshi, Aedes (Neomelaniconion) circumluteolus, Aedes (Aedimorphus) durbanensis, and Culex (Lasioconops) poicilipes may be involved. The aim of the study was to determine the vertebrate blood meal sources of potential RVFV mosquito vectors in north-eastern KZN and to characterize the host-biting network. METHODS: Blood-fed mosquitoes were collected monthly from November 2019 to February 2023 using a backpack aspirator, CO2-baited Centers for Disease Control and Prevention (CDC) miniature light traps and tent traps, in the vicinity of water bodies and livestock farming households. The mosquitoes were morphologically identified. DNA was extracted from individual mosquitoes and used as templates to amplify the vertebrate cytochrome c oxidase I (COI) and cytochrome b (cytb) genes using conventional polymerase chain reaction (PCR). Amplicons were sequenced and queried in GenBank and the Barcode of Life Data systems to identify the vertebrate blood meal sources and confirm mosquito identifications. All mosquitoes were screened for RVFV using real time reverse transcription (RT)-PCR. RESULTS: We identified the mammalian (88.8%) and avian (11.3%) blood meal sources from 409 blood-fed mosquitoes. Aedes circumluteolus (n = 128) made up the largest proportion of collected mosquitoes. Cattle (n = 195) and nyala (n = 61) were the most frequent domestic and wild hosts, respectively. Bipartite network analysis showed that the rural network consisted of more host-biting interactions than the reserve network. All mosquitoes tested negative for RVFV. CONCLUSIONS: Several mosquito species, including Ae. circumluteolus, and vertebrate host species, including cattle and nyala, could play a central role in RVFV transmission. Future research in this region should focus on these species to better understand RVFV amplification.

Is Culex modestus a New Usutu virus vector?

Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.

Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

Culex-Transmitted Diseases: Mechanisms, Impact, and Future Control Strategies using Wolbachia.

Mosquitoes of the Culex genus are responsible for a large burden of zoonotic virus transmission globally. Collectively, they play a significant role in the transmission of medically significant diseases such as Japanese encephalitis virus and West Nile virus. Climate change, global trade, habitat transformation and increased urbanisation are leading to the establishment of Culex mosquitoes in new geographical regions. These novel mosquito incursions are intensifying concerns about the emergence of Culex-transmitted diseases and outbreaks in previously unaffected areas. New mosquito control methods are currently being developed and deployed globally. Understanding the complex interaction between pathogens and mosquitoes is essential for developing new control strategies for Culex species mosquitoes. This article reviews the role of Culex mosquitos as vectors of zoonotic disease, discussing the transmission of viruses across different species, and the potential use of Wolbachia technologies to control disease spread. By leveraging the insights gained from recent successful field trials of Wolbachia against Aedes-borne diseases, we comprehensively discuss the feasibility of using this technique to control Culex mosquitoes and the potential for the development of next generational Wolbachia-based control methods.

An amplicon-based sequencing approach for Usutu virus characterization.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus.

Determination of Species Identity and Genetic Diversity of Aedes aegypti and Other Medically Important Mosquitoes of India Using DNA Barcoding.

Aedes aegypti-borne viruses (i.e., dengue, chikungunya, and Zika) have become endemic to India, posing a severe threat to public health. Vector control remains the mainstay of disease management due to nonavailability of licensed vaccines/therapeutics. Conventional morpho-taxonomical methods cannot differentiate between closely related sibling species or species complexes, and hence we evaluated two molecular markers, mitochondrial cytochrome c oxidase subunit 1 (Cox1) and nuclear DNA internal transcribed spacer 2 (-2) gene sequences, to characterize seven populations of Ae. aegypti and four medically important mosquito species (Aedes albopictus, Anopheles stephensi, Culex tritaeniorhyncus, and Culex murrelli). DNA extracted from the 11 mosquito populations (two mosquitoes per population) was polymerase chain reaction amplified, sequenced, and analyzed. Molecular characterization was found to be congruent with morphological identification, suggesting no variants or cryptic species exist in Ae. aegypti and the other mosquitoes studied. Phylogenetic analysis with sequences obtained with Cox1 gene of Ae. aegypti and other Aedes and non-Aedes mosquito species showed clustering of sequences from different species representing different clades, distinctly separating one taxon from the other, whereas ITS-2 sequences of Aedes aegypti from across the world clustered tightly. Nucleotide divergence values revealed a low percentage of intraspecies variation and a higher percentage of interspecies variation. The present study authenticates the applicability of Cox1 and ITS-2 in the precise identification of Ae. aegypti mosquitoes against cryptic or sibling species. Cox1 appeared to be a more reliable marker because it showed distinct clustering of mosquito species, and some sequence variations to represent genetic diversity.

Disclosing the virome of Aedes, Anopheles and Culex female mosquitoes, Alto Pantanal of Mato Grosso, Brazil, 2019.

Using Illumina NextSeq sequencing and bioinformatics, we identified and characterized thirty-three viral sequences of unsegmented and multipartite viral families in Aedes spp., Culex sp. and Anopheles darlingi female mosquito pools from Porto São Luiz and Pirizal, Alto Pantanal. Seventeen sequences belong to unsegmented viral families, twelve represent putative novel insect-specific viruses (ISVs) within families Chuviridae (3/33; partial genomes) and coding-complete sequences of Xinmoviridae (1/33), Rhabdoviridae (2/33) and Metaviridae (6/33); and five coding-complete sequences of already-known ISVs. Notably, two putative novel rhabdoviruses, Corixo rhabdovirus 1 and 2, were phylogenetically related to Coxipo dielmovirus, but separated from other Alpharhabdovirinae genera, sharing Anopheles spp. as host. Regarding multipartite families, sixteen segments of different putative novel viruses were identified (13 coding-complete segments) within Durnavirales (4/33), Elliovirales (1/33), Hareavirales (3/33) and Reovirales (8/33) orders. Overall, this study describes twenty-eight (28/33) putative novel ISVs and five (5/33) already described viruses using metagenomics approach.

Study on the temporal and spatial distribution of Culex mosquitoes in Hanoi, Vietnam.

Arboviruses transmitted by mosquitoes, including Japanese encephalitis virus (JEV), present a substantial global health threat. JEV is transmitted by mosquitoes in the genus Culex, which are common in both urban and rural areas in Vietnam. In 2020, we conducted a 1-year survey of Culex mosquito abundance in urban, suburban, and peri-urban areas of Hanoi using CDC-light traps. Mosquitoes were identified to species and sorted into pools based on species, sex, and trap location. The mosquito pools were also investigated by RT-qPCR for detection of JEV. In total, 4829 mosquitoes were collected over a total of 455 trap-nights, across 13 months. Collected mosquitoes included Culex, Aedes, Anopheles, and Mansonia species. Culex mosquitoes, primarily Cx. quinquefasciatus, predominated, especially in peri-urban areas. Most Culex mosquitoes were caught in the early months of the year. The distribution and abundance of mosquitoes exhibited variations across urban, suburban, and peri-urban sites, emphasizing the influence of environmental factors such as degree of urbanization, temperature and humidity on Culex abundance. No JEV was detected in the mosquito pools. This study establishes baseline knowledge of Culex abundance and temporal variation, which is crucial for understanding the potential for JEV transmission in Hanoi.

Ecology and geography of Cache Valley virus assessed using ecological niche modeling.

BACKGROUND: Cache Valley virus (CVV) is an understudied Orthobunyavirus with a high spillover transmission potential due to its wide geographical distribution and large number of associated hosts and vectors. Although CVV is known to be widely distributed throughout North America, no studies have explored its geography or employed computational methods to explore the mammal and mosquito species likely participating in the CVV sylvatic cycle. METHODS: We used a literature review and online databases to compile locality data for CVV and its potential vectors and hosts. We linked location data points with climatic data via ecological niche modeling to estimate the geographical range of CVV and hotspots of transmission risk. We used background similarity tests to identify likely CVV mosquito vectors and mammal hosts to detect ecological signals from CVV sylvatic transmission. RESULTS: CVV distribution maps revealed a widespread potential viral occurrence throughout North America. Ecological niche models identified areas with climate, vectors, and hosts suitable to maintain CVV transmission. Our background similarity tests identified Aedes vexans, Culiseta inornata, and Culex tarsalis as the most likely vectors and Odocoileus virginianus (white-tailed deer) as the most likely host sustaining sylvatic transmission. CONCLUSIONS: CVV has a continental-level, widespread transmission potential. Large areas of North America have suitable climate, vectors, and hosts for CVV emergence, establishment, and spread. We identified geographical hotspots that have no confirmed CVV reports to date and, in view of CVV misdiagnosis or underreporting, can guide future surveillance to specific localities and species.

Virulence and transmission vary between Usutu virus lineages in Culex pipiens.

Usutu virus (USUV) is a zoonotic arbovirus infecting mainly wild birds. It is transmitted by ornithophilic mosquitoes, mainly of the genus Culex from birds to birds and to several vertebrate dead-end hosts. Several USUV lineages, differing in their virulence have emerged in the last decades and now co-circulate in Europe, impacting human populations. However, their relative transmission and effects on their mosquito vectors is still not known. We thus compared the vector competence and survival of Culex pipiens mosquitoes experimentally infected with two distinct USUV lineages, EU2 and EU3, that are known to differ in their virulence and replication in vertebrate hosts. Infection rate was variable among blood feeding assays but variations between EU2 and EU3 lineages were consistent suggesting that Culex pipiens was equally susceptible to infection by both lineages. However, EU3 viral load increased with viral titer in the blood meal while EU2 viral load was high at all titers which suggest a greater replication of EU2 than EU3 in mosquito. While their relative transmission efficiencies are similar, at least at low blood meal titer, positive correlation between transmission and blood meal titer was observed for EU3 only. Contrary to published results in vertebrates, EU3 induced a higher mortality to mosquitoes (i.e. virulence) than EU2 whatever the blood meal titer. Therefore, we found evidence of lineage-specific differences in vectorial capacity and virulence to both the vector and vertebrate host which lead to balanced propagation of both viral lineages. These results highlight the need to decipher the interactions between vectors, vertebrate hosts, and the diversity of arbovirus lineages to fully understand transmission dynamics.

Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus.

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti-field), RecL (Ae. aegypti-laboratory) and CqSLab (Cx. quinquefasciatus-laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission.

Evaluation of the vector competence for Batai virus of native Culex and exotic Aedes species in Central Europe.

BACKGROUND: Batai virus (BATV) is a zoonotic arbovirus of veterinary importance. A high seroprevalence in cows, sheep and goats and infection in different mosquito species has been observed in Central Europe. Therefore, we studied indigenous as well as exotic species of the genera Culex and Aedes for BATV vector competence at different fluctuating temperature profiles. METHODS: Field caught Culex pipiens biotype pipiens, Culex torrentium, Aedes albopictus and Aedes japonicus japonicus from Germany and Aedes aegypti laboratory colony were infected with BATV strain 53.3 using artificial blood meals. Engorged mosquitoes were kept under four (Culex species) or three (Aedes species) fluctuating temperature profiles (18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C) at a humidity of 70% and a dark/light rhythm of 12:12 for 14 days. Transmission was measured by testing the saliva obtained by forced salivation assay for viable BATV particles. Infection rates were analysed by testing whole mosquitoes for BATV RNA by quantitative reverse transcription PCR. RESULTS: No transmission was detected for Ae. aegypti, Ae. albopictus or Ae. japonicus japonicus. Infection was observed for Cx. p. pipiens, but only in the three conditions with the highest temperatures (21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C). In Cx. torrentium infection was measured at all tested temperatures with higher infection rates compared with Cx. p. pipiens. Transmission was only detected for Cx. torrentium exclusively at the highest temperature of 27 ± 5 °C. CONCLUSIONS: Within the tested mosquito species, only Cx. torrentium seems to be able to transmit BATV if the climatic conditions are feasible.

Broadscale spatial synchrony in a West Nile virus mosquito vector across multiple timescales.

Insects often exhibit irruptive population dynamics determined by environmental conditions. We examine if populations of the Culex tarsalis mosquito, a West Nile virus (WNV) vector, fluctuate synchronously over broad spatial extents and multiple timescales and whether climate drives synchrony in Cx. tarsalis, especially at annual timescales, due to the synchronous influence of temperature, precipitation, and/or humidity. We leveraged mosquito collections across 9 National Ecological Observatory Network (NEON) sites distributed in the interior West and Great Plains region USA over a 45-month period, and associated gridMET climate data. We utilized wavelet phasor mean fields and wavelet linear models to quantify spatial synchrony for mosquitoes and climate and to calculate the importance of climate in explaining Cx. tarsalis synchrony. We also tested whether the strength of spatial synchrony may vary directionally across years. We found significant annual synchrony in Cx. tarsalis, and short-term synchrony during a single period in 2018. Mean minimum temperature was a significant predictor of annual Cx. tarsalis spatial synchrony, and we found a marginally significant decrease in annual Cx. tarsalis synchrony. Significant Cx. tarsalis synchrony during 2018 coincided with an anomalous increase in precipitation. This work provides a valuable step toward understanding broadscale synchrony in a WNV vector.

Vegetation structure drives mosquito community composition in UK's largest managed lowland wetland.

PURPOSE: The rising burden of mosquito-borne diseases in Europe extends beyond urban areas, encompassing rural and semi-urban regions near managed and natural wetlands evidenced by recent outbreaks of Usutu and West Nile viruses. While wetland management policies focus on biodiversity and ecosystem services, few studies explore the impact on mosquito vectors. METHODS: Our research addresses this gap, examining juvenile mosquito and aquatic predator communities in 67 ditch sites within a South England coastal marsh subjected to different wetland management tiers. Using joint distribution models, we analyse how mosquito communities respond to abiotic and biotic factors influenced by wetland management. RESULTS: Of the 12 mosquito species identified, Culiseta annulata (Usutu virus vector) and Culex pipiens (Usutu and West Nile virus vector) constitute 47% of 6825 larval mosquitoes. Abundant predators include Coleoptera (water beetles) adults, Corixidae (water boatmen) and Zygoptera (Damselfy) larvae. Models reveal that tier 3 management sites (higher winter water levels, lower agricultural intensity) associated with shade and less floating vegetation are preferred by specific mosquito species. All mosquito species except Anopheles maculipennis s.l., are negatively impacted by potential predators. Culiseta annulata shows positive associations with shaded and turbid water, contrary to preferences of Corixidae predators. CONCLUSIONS: Tier 3 areas managed for biodiversity, characterised by higher seasonal water levels and reduced livestock grazing intensity, provide favourable habitats for key mosquito species that are known vectors of arboviruses, such as Usutu and West Nile. Our findings emphasise the impact of biodiversity-focused wetland management, altering mosquito breeding site vegetation to enhance vector suitability. Further exploration of these trade-offs is crucial for comprehending the broader implications of wetland management.

Mayaro virus, a potential threat for Europe: vector competence of autochthonous vector species.

BACKGROUND: Mayaro virus (MAYV) is an emerging alphavirus, primarily transmitted by the mosquito Haemagogus janthinomys in Central and South America. However, recent studies have shown that Aedes aegypti, Aedes albopictus and various Anopheles mosquitoes can also transmit the virus under laboratory conditions. MAYV causes sporadic outbreaks across the South American region, particularly in areas near forests. Recently, cases have been reported in European and North American travelers returning from endemic areas, raising concerns about potential introductions into new regions. This study aims to assess the vector competence of three potential vectors for MAYV present in Europe. METHODS: Aedes albopictus from Italy, Anopheles atroparvus from Spain and Culex pipiens biotype molestus from Belgium were exposed to MAYV and maintained under controlled environmental conditions. Saliva was collected through a salivation assay at 7 and 14 days post-infection (dpi), followed by vector dissection. Viral titers were determined using focus forming assays, and infection rates, dissemination rates, and transmission efficiency were calculated. RESULTS: Results indicate that Ae. albopictus and An. atroparvus from Italy and Spain, respectively, are competent vectors for MAYV, with transmission possible starting from 7 dpi under laboratory conditions. In contrast, Cx. pipiens bioform molestus was unable to support MAYV infection, indicating its inability to contribute to the transmission cycle. CONCLUSIONS: In the event of accidental MAYV introduction in European territories, autochthonous outbreaks could potentially be sustained by two European species: Ae. albopictus and An. atroparvus. Entomological surveillance should also consider certain Anopheles species when monitoring MAYV transmission.

Demographic inference from the mt-DNA COI gene and wing geometry of Culex gelidus (Diptera: Culicidae), an important vector of Japanese encephalitis in Thailand.

Culex gelidus (Diptera: Culicidae), an important vector of the Japanese encephalitis virus (JEV), contributes to human viral encephalitis in many Asian countries, including Thailand. This study represents the first investigation of the demographic patterns of Cx. gelidus populations in Thailand using cytochrome c oxidase subunit I (COI) gene analysis and wing geometric morphometrics (GM). Mosquitoes were collected from 10 provinces across six regions of Thailand in 2022. Analysis of the COI sequences (n = 182) indicated high haplotype diversity (0.882) and low nucleotide diversity (0.006), with 72 haplotypes identified. The haplotype network demonstrated no profound splits among the geographic populations. Neutral tests, including Tajima's D and Fu's Fs, displayed negative values, with a significant result observed for Fu's Fs (-33.048, p < 0.05). The mismatch distribution analysis indicated that the population does not statistically deviate from a model of sudden population expansion (SSD = 0.010, p > 0.05; Rg = 0.022, p > 0.05). The estimations suggest that the Cx. gelidus population in Thailand began its expansion approximately between 459,243 and 707,011 years ago. The Mantel test showed no significant relationship between genetic and geographic distances (r = 0.048, p > 0.05). Significant phenotypic differences (based on wing shape) were observed among most populations. Additionally, in this study, we found no significant relationships between phenotypic and genetic distances (r = 0.250, p > 0.05). Understanding the genetic and morphological dynamics of Cx. gelidus is vital for developing targeted surveillance and vector control measures. This knowledge will also help to predict how future environmental changes might affect these populations, thereby informing long-term vector management strategies.

Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome.

In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex.IMPORTANCECulex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila.

BACKGROUND: Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. METHODS: Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. RESULTS: An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. CONCLUSIONS: Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.