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Microculex is a subgenus of Culex that is mainly associated with natural breeding sites in wild environments such as cut bamboos, tree holes and bromeliads. However, recent findings of Culex (Microculex) species in bromeliads in urban areas and in artificial breeding sites close to human habitations suggest that they are becoming more tolerant of urbanization. Whilst dispersion studies of this subgenus have shown the ecological valency of some species in relation to human-impacted areas, there is a scarcity of studies on the blood-feeding habits, vector capacity and taxonomy of Microculex. As all the information about this subgenus is fragmented and restricted in most cases to physical libraries, this review seeks to compile all the information on Microculex published over the last century and a half so that this can be more easily consulted by specialists interested in the subject. The ability of some species of this subgenus to adapt to human-impacted environments by colonizing artificial breeding sites and thus become a potential vector of animal and human diseases is also discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Disease-vector mosquito monitoring is an essential prerequisite to optimize control interventions and evidence-based risk predictions. However, conventional entomological monitoring methods are labor- and time-consuming and do not allow high temporal/spatial resolution. In 2022, a novel system coupling an optical sensor with machine learning technologies (VECTRACK) proved effective in counting and identifying Aedes albopictus and Culex pipiens adult females and males. Here, we carried out the first extensive field evaluation of the VECTRACK system to assess: (i) whether the catching capacity of a commercial BG-Mosquitaire trap (BGM) for adult mosquito equipped with VECTRACK (BGM + VECT) was affected by the sensor; (ii) the accuracy of the VECTRACK algorithm in correctly classifying the target mosquito species genus and sex; (iii) Ae. albopictus capture rate of BGM with or without VECTRACK., Methods: The same experimental design was implemented in four areas in northern (Bergamo and Padua districts), central (Rome) and southern (Procida Island, Naples) Italy. In each area, three types of traps-one BGM, one BGM + VECT and the combination of four sticky traps (STs)-were rotated each 48 h in three different sites. Each sampling scheme was replicated three times/area. Collected mosquitoes were counted and identified by both the VECTRACK algorithm and operator-mediated morphological examination. The performance of the VECTRACK system was assessed by generalized linear mixed and linear regression models. Aedes albopictus capture rates of BGMs were calculated based on the known capture rate of ST., Results: A total of 3829 mosquitoes (90.2% Ae. albopictus) were captured in 18 collection-days/trap/site. BGM and BGM + VECT showed a similar performance in collecting target mosquitoes. Results show high correlation between visual and automatic identification methods (Spearman Ae. albopictus: females = 0.97; males = 0.89; P < 0.0001) and low count errors. Moreover, the results allowed quantifying the heterogeneous effectiveness associated with different trap types in collecting Ae. albopictus and predicting estimates of its absolute density., Conclusions: Obtained results strongly support the VECTRACK system as a powerful tool for mosquito monitoring and research, and its applicability over a range of ecological conditions, accounting for its high potential for continuous monitoring with minimal human effort., (© 2024. The Author(s).)

Background: The recent rise in the transmission of mosquito-borne diseases such as dengue virus (DENV), Zika (ZIKV), chikungunya (CHIKV), Oropouche (OROV), and West Nile (WNV) is a major concern for public health managers worldwide. Emerging technologies for automated remote mosquito classification can be supplemented to improve surveillance systems and provide valuable information regarding mosquito vector catches in real time., Methods: We coupled an optical sensor to the entrance of a standard mosquito suction trap (BG-Mosquitaire) to record 9151 insect flights in two Brazilian cities: Rio de Janeiro and Brasilia. The traps and sensors remained in the field for approximately 1 year. A total of 1383 mosquito flights were recorded from the target species: Aedes aegypti and Culex quinquefasciatus. Mosquito classification was based on previous models developed and trained using European populations of Aedes albopictus and Culex pipiens., Results: The VECTRACK sensor was able to discriminate the target mosquitoes (Aedes and Culex genera) from non-target insects with an accuracy of 99.8%. Considering only mosquito vectors, the classification between Aedes and Culex achieved an accuracy of 93.7%. The sex classification worked better for Cx. quinquefasciatus (accuracy: 95%; specificity: 95.3%) than for Ae. aegypti (accuracy: 92.1%; specificity: 88.4%)., Conclusions: The data reported herein show high accuracy, sensitivity, specificity and precision of an automated optical sensor in classifying target mosquito species, genus and sex. Similar results were obtained in two different Brazilian cities, suggesting high reliability of our findings. Surprisingly, the model developed for European populations of Ae. albopictus worked well for Brazilian Ae. aegypti populations, and the model developed and trained for Cx. pipiens was able to classify Brazilian Cx. quinquefasciatus populations. Our findings suggest this optical sensor can be integrated into mosquito surveillance methods and generate accurate automatic real-time monitoring of medically relevant mosquito species., (© 2024. The Author(s).)

Background: Antananarivo, the capital city of Madagascar, is experiencing a steady increase in population growth. Due to the abundance of mosquito vectors in this locality, the population exposed to mosquito-borne diseases is therefore also increasing, as is the risk of epidemic episodes. The aim of the present study was to assess, in a resource-limited setting, the information on mosquito population dynamics and disease transmission risk that can be provided through a longitudinal entomological study carried out in a multi-host single site., Methods: Mosquitoes were collected every 15 days over 16 months (from January 2017 to April 2018) using six CDC-light traps in a peri-urban area of Antananarivo. Multivariable generalised linear models were developed using indoor and outdoor densities of the predominant mosquito species as response variables and moon illumination, environmental data and climatic data as the explanatory variables., Results: Overall, 46,737 mosquitoes belonging to at least 20 species were collected, of which Culex antennatus (68.9%), Culex quinquefasciatus (19.8%), Culex poicilipes (3.7%) and Anopheles gambiae sensu lato (2.3%) were the most abundant species. Mosquito densities were observed to be driven by moon illumination and climatic factors interacting at different lag periods. The outdoor models demonstrated biweekly and seasonal patterns of mosquito densities, while the indoor models demonstrated only a seasonal pattern., Conclusions: An important diversity of mosquitoes exists in the peri-urban area of Antananarivo. Some well-known vector species, such as Cx. antennatus, a major vector of West Nile virus (WNV) and Rift-Valley fever virus (RVFV), Cx. quinquefasciatus, a major vector of WNV, Cx. poicilipes, a candidate vector of RVFV and An. gambiae sensu lato, a major vector of Plasmodium spp., are abundant. Importantly, these four mosquito species are present all year round, even though their abundance declines during the cold dry season, with the exception of Cx. quinquefasciatus. The main drivers of their abundance were found to be temperature, relative humidity and precipitation, as well as-for outdoor abundance only-moon illumination. Identifying these drivers is a first step towards the development of pathogen transmission models (R0 models), which are key to inform public health stakeholders on the periods of most risk for vector-borne diseases., (© 2024. The Author(s).)

Classification of mosquitoes with overlapping features remains problematic when using traditional morphological identification alone. In this study, we used molecular methods to elucidate the taxonomic status of Culex tritaeniorhynchus, Culex annulus, and Culex pseudovishnui species as vectors of the Japanese encephalitis virus belonging to the Culex vishnui subgroup and gene flow among them. In this study, 76, 59, and 3 samples of Cx. annulus, Cx. tritaeniorhynchus, and Cx. pseudovishnui, respectively, were collected around Taiwan. Phylogenetic analysis and genetic divergence were based on genomic sequence variations in ribosomal DNA and the internal transcribed spacer (rDNA) and cytochrome c oxidase subunit I (COI). Our results revealed that Cx. annulus and Cx. vishnui are genetically similar and share a gene pool among the species from Taiwan and other Asian countries. However, two hidden taxa of Cx. tritaeniorhynchus, which clustered together according to the rDNA sequences, were discovered based on the COI sequences. In addition, Cx. pseudovishnui has different gene pools from those of the strains from other countries, implying that the population from Taiwan is probably either a unique strain or a sibling species. This study provides molecular information on the taxonomic status of the species in the Cx. vishnui subgroup in Taiwan and gene flow between these species, providing valuable information for vector control operations and the delineation of the evolutionary process.

Mosquitoes are medically important insects, and accurate species identification is crucial to understanding vector biology, forming the cornerstone of successful vector control programs. Identification is difficult owing to morphologically similar species. Wing morphometrics can provide a simple, fast, and accurate way to classify species, and using it as a method to differentiate vector species among its cryptic congeners has been underexplored. Using a total of 227 mosquitoes and 20 landmarks per specimen, we demonstrated the utility of wing morphometrics in differentiating species two groups occurring in sympatry - Culex (Culex) vishnui group and Culex (Lophoceraomyia) subgenus, as well as explored population-level variation in the wing shape of Aedes albopictus across habitats. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Procrustes ANOVA regression and CVA based on wing shape reflected that the wing landmarks across all species differed significantly, and leave-one-out cross validation revealed an overall high accuracy of >97% for the two Culex groups. Wing morphometrics uncovered population-level variation within Aedes albopictus, but cross validation accuracy was low. Overall, we show that wing geomorphometric analysis is able to resolve cryptic Culex species (including vectors) occurring sympatrically, and is a robust tool for identifying mosquitoes reliably., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)

Background: Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females., Methods: We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search., Results: In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns., Conclusions: In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors., (© 2024. The Author(s).)

Mosquitoes (Diptera: Culicidae) pose a significant threat to public health worldwide, especially in tropical and subtropical regions, where they act as primary vectors in transmission of infectious agents. In Peru, 182 culicid species have been identified and several species of the genus Culex are known to transmit arboviruses. However, knowledge of mosquito diversity and distribution remains limited, with many studies focusing on specific regions only. Here, we describe a new morphological variation of Cx. (Culex) coronator Dyar and Knab, 1906, and report the presence of Culex (Carrollia) bonnei Dyar, 1921 in the central region of Peru, Huanuco. Specimens were obtained through larvae collections and identified through morphologic characterization, including dissection of male genitalia, and molecular analyses. In total, 17 mosquitoes were analyzed, and the genitalia of the male specimens allowed the identification of Cx. coronator and Cx. bonnei. Partial sequences of the CoxI gene corresponding to these two species were obtained (N = 10). Phylogenetic analysis revealed that the sequences of Cx. coronator grouped in a monophyletic clade with sequences ascribed to other species corresponding to the subgenus Carrollia, while Cx. bonnei specimens formed a monophyletic clade with homologous sequences from GenBank. This study underscores the importance of continued efforts to study the diversity and distribution of mosquitoes in Peru, including their potential role as vectors of human pathogens, to underpin effective disease control and prevention strategies, highlighting the importance of a complemented morphological and molecular analysis., (© 2024. The Author(s).)

Background: Analyses of the temporal distribution of mosquitoes are presented in statistical charts, but it is difficult to prove in statistics whether differences in peak periods exist among different years or habitats. This study aimed to investigate the application of circular statistics in determining the peak period and a comparison of differences., Methods: Surveillance of adult mosquitoes was conducted twice a month by light traps in five different habitats from March to November for 3 years (2021-2023) in Qingdao, Shandong Province, China. The Kruskal-Wallis test was performed to determine the differences in mosquito density among different years and habitats. Circular statistics and line charts were employed to determine the peak period and a comparison of differences., Results: Among a total of 14,834 adult mosquitoes comprising five mosquito species from four genera, Culex pipiens pallens was dominant and accounted for 89.6% of the specimens identified. Aedes albopictus, Armigeres subalbatus, and Anopheles sinensis made up 5.7%, 4.2%, and 0.5%, respectively. Culex tritaeniorhynchus accounted for less than 0.1%. The mean mosquito density (females/trap night) for the trapping period was 10.3 in 2021, 5.6 in 2022, and 3.6 in 2023. Among five habitats, the highest mosquito density was 8.9 in livestock sheds, followed by 6.8 in parks, 5.9 in rural dwellings, 5.5 in urban dwellings, and 5.4 in hospitals. No statistically significant differences were found among different years (H = 1.96, d.f. 2, P = 0.376) and habitats (H = 0.45, d.f. 4, P = 0.978). Overall, the peak period of mosquito activity fell in the months from June to September. The peak period among 3 years differed significantly (F (2,7022)  = 119.17, P < 0.01), but there were no statistically significant differences in peak period among different habitats (F (4,7020)  = -159.09, P > 0.05)., Conclusion: Circular statistics could be effectively combined with statistical charts to elucidate the peak period of mosquitoes and determine the differences in statistics among different years and habitats. These findings will provide valuable information for mosquito control and public health management., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s).)

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., (© 2024. Crown.)

Mosquito-borne diseases have emerged in North Borneo in Malaysia due to rapid changes in the forest landscape, and mosquito surveillance is key to understanding disease transmission. However, surveillance programmes involving sampling and taxonomic identification require well-trained personnel, are time-consuming and labour-intensive. In this study, we aim to use a deep leaning model (DL) to develop an application capable of automatically detecting mosquito vectors collected from urban and suburban areas in North Borneo, Malaysia. Specifically, a DL model called MobileNetV2 was developed using a total of 4880 images of Aedes aegypti , Aedes albopictus and Culex quinquefasciatus mosquitoes, which are widely distributed in Malaysia. More importantly, the model was deployed as an application that can be used in the field. The model was fine-tuned with hyperparameters of learning rate 0.0001, 0.0005, 0.001, 0.01 and the performance of the model was tested for accuracy, precision, recall and F1 score. Inference time was also considered during development to assess the feasibility of the model as an app in the real world. The model showed an accuracy of at least 97%, a precision of 96% and a recall of 97% on the test set. When used as an app in the field to detect mosquitoes with the elements of different background environments, the model was able to achieve an accuracy of 76% with an inference time of 47.33 ms. Our result demonstrates the practicality of computer vision and DL in the real world of vector and pest surveillance programmes. In the future, more image data and robust DL architecture can be explored to improve the prediction result.

Background: Culex pipiens quinquefasciatus Say (Cx. quinquefasciatus) and Culex pipiens form molestus Forskal (Cx. molestus) in the Culex pipiens complex group show considerable differences in host seeking, blood feeding, mating behavior and in vector competence. Blood-feeding mosquito behaviors are closely related to their olfactory gene expression and olfactory gene repertoire composition. Comparing olfactory genes between these two subspecies with significantly different blood-feeding behaviors can support further research on the molecular mechanism of the Culex pipiens complex olfactory sensory system, providing a new approach for determining candidate attractant or repellent compounds., Methods: Non-blood-feeding (NBF) and post-blood-feeding (PBF) olfactory system transcriptomes of the two subspecies were sequenced, and the biological functions of their differentially expressed genes were described by bioinformatics analysis. A quantitative polymerase chain reaction (qPCR) was applied to validate the RNA-seq data. The roles of particular olfactory receptors in Cx. quinquefasciatus blood-feeding behaviors were evaluated by RNAi., Results: Five, 7, 24, and 3 Cx. quinquefasciatus-specific OBPs, Cx. molestus-specific OBPs, Cx. quinquefasciatus-specific ORs and Cx. molestus-specific ORs were identified, respectively. The majority of selected ORs were consistent with the predicted transcriptome sequencing results after qRT-PCR validation. OR5 was expressed only in Cx. quinquefasciatus, and OR65 was the only gene upregulated after blood feeding in Cx. molestus. The blood-feeding rates of the OR5 and OR78 dsRNA groups were significantly lower (4.3%±3.1% and 13.3%±11.5%) than those of the enhanced green fluorescence protein (EGFP) group (64.5%±8.7%)., Conclusion: Most OBPs and ORs were expressed in both subspecies but showed divergence in expression level. OR5 and OR65 might be species-specific expressed genes that regulate the olfactory behaviors of Cx. quinquefasciatus and Cx. molestus, respectively. The RNA interference of OR5 and OR78 could inhibit the blood-feeding behavior of Cx. quinquefasciatus, providing new targets for screening effective repellent compounds to control mosquito-borne diseases effectively and efficiently., Competing Interests: The authors have declared that no competing interests exist.

Southern Russia remains affected by West Nile virus (WNV). In the current study, we identified the spatial determinants of WNV distribution in an area with endemic virus transmission, with special reference to the urban settings, by mapping probable points of human infection acquisition and points of virus detection in mosquitoes, ticks, birds, and mammals during 1999-2016. The suitability of thermal conditions for extrinsic virus replication was assessed based on the approach of degree-day summation and their changes were estimated by linear trend analysis. A generalized linear model was used to analyze the year-to-year variation of human cases versus thermal conditions. Environmental suitability was determined by ecological niche modelling using MaxEnt software. Human population density was used as an offset to correct for possible bias. Spatial analysis of virus detection in the environment showed significant contributions from surface temperature, altitude, and distance from water bodies. When indicators of location and mobility of the human population were included, the relative impact of factors changed, with roads becoming most important. When the points of probable human case infection were added, the percentage of leading factors changed only slightly. The urban environment significantly increased the epidemic potential of the territory and created quite favorable conditions for virus circulation. The private building sector with low-storey houses and garden plots located in the suburbs provided a connection between urban and rural transmission cycles., Competing Interests: The authors have declared that no competing interests exist.

Background: Odorant-binding proteins (OBPs) play important roles in many physiological processes of mosquitoes. Previous high-throughput sequencing studies have revealed that some OBPs of Culex quinquefasciatus might be involved in the development of resistance to insecticides., Methods: Based on the results of sequencing analyses, the OBP28 gene was selected for evaluation in this study. Three laboratory strains of Cx. quinquefasciatus [susceptible strain (SS), deltamethrin-resistant strain 1 (HN) and deltamethrin-resistant strain 2 (RR)] were first examined by using the Centers for Disease Control and Prevention bottle bioassay, after which the expression level of the OBP28 gene in the susceptible and deltamethrin-resistant strains was determined by real-time quantitative polymerase chain reaction. The OBP28 gene in deltamethrin-resistant strain RR was silenced using RNA interference technology. The expression level of OBP28 and the resistance level were tested in the silenced strain and control strain after microinjection of double-stranded RNA for a 48-h interference period. Four field-collected strains (henceforth 'field strains') of Cx. quinquefasciatus were also examined for their resistance to deltamethrin and levels of OBP28 expression. Finally, a correlation analysis between deltamethrin resistance and gene expression was carried out for all seven strains, i.e. the four field strains and the three laboratory strains., Results: In the bioassay, the mortality of SS, HN and RR was 100%, 21.33% and 1.67%, respectively. The relative expression levels of OBP28 in strains HN and RR were 6.30- and 6.86-fold higher, respectively, than that of strain SS. After silencing of the OBP28 gene, the mortality of strain RR was 72.20% and that of the control strain 26.32%. The mortality of strain RR increased significantly after interference compared to that of the control strain. There was a negative correlation between OBP28 gene expression and mortality in adult mosquitoes after exposure to deltamethrin., Conclusions: To our knowledge, this study shows for the first time a correlation between the expression of a gene coding for OBP and insecticide resistance in mosquitoes. The potential resistance mechanism that was elucidated provides a new target gene for the surveillance of resistance in mosquitoes., (© 2021. The Author(s).)

Culex longitubus Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex. The larva is most similar to the larva of Cx. tianpingensis Chen from China, but is distinguished by the length of the siphon and the anal papillae, the form of the comb scales and pecten spines, and the development of setae 7-P, 13-T, 1-X, and 4-X. The adults have wing markings and male genitalia similar to those of species of the Mimeticus Complex. Phylogenetic analysis of COI sequences revealed that the new species is closely related to Cx. murrelli Lien of the Mimulus Complex. The immature stages of the new species were found in stagnant pools and marshes at high altitudes in several districts of Bhutan., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Culex quinquefasciatus is not only a biting nuisance but also an important vector of fatal diseases. In Saudi Arabia, management measures to control this mosquito and thereby prevent associated disease transmission have focused on insecticides. Nevertheless, information on the resistance status of C. quinquefasciatus is insufficient, especially concerning insecticides containing novel classes of insecticides., Methods: We evaluated six insecticides belonging to four classes of insecticides (insect growth regulators [3], avermectins [1], diamides [1] and spinosyns [1]) for toxicity and resistance in eight C. quinquefasciatus populations (from Ishbiliya, Al-Masfa, Al-Masanie, Al-Washlah, Al-Nakhil, Irqah, Al-Suwaidi and Al-Ghanemiya) following World Health Organisation protocols., Results: Resistance status ranging from susceptibility/low resistance to high resistance, in comparison with the susceptible strain, was detected for cyromazine in the eight C. quinquefasciatus populations: Ishbiliya (resistance ratio [RR] = 3.33), Al-Masfa (RR = 4.33), Al-Masanie (RR = 3.67), Al-Washlah (RR = 2.33), Al-Nakhil (RR = 5.33), Irqah (RR = 7.00), Al-Suwaidi (RR = 21.33) and Al-Ghanemiya (RR = 16.00). All C. quinquefasciatus populations exhibited a high level of resistance to diflubenzuron (RR = 13.33-43.33), with the exception of Al-Nakhil which exhibited moderate resistance (RR = 10.00). Susceptibility/low resistance to high resistance was observed for triflumuron in the eight C. quinquefasciatus populations: Ishbiliya (RR = 0.50), Al-Ghanemiya (RR =  3.00), Al-Suwaidi (RR =  10.00), Al-Masfa (RR =  5.00), Al-Masanie (RR =  10.00), Al-Nakhil (RR =  5.00), Irqah (RR =  5.00) and Al-Washlah (RR =  15.00). Susceptibility/low resistance was assessed for abamectin, chlorantraniliprole and spinosad in all C. quinquefasciatus populations, with RR ranges of 0.25-3.50, 0.17-2.19, and 0.02-0.50, respectively. However, the population collected from Irqah showed high resistance to chlorantraniliprole (RR = 11.93)., Conclusions: The detection of widespread resistance to insect growth regulators in C. quinquefasciatus highlights an urgent need to establish integrated vector management strategies. Our results may facilitate the selection of potent insecticides for integrated vector management programmes for C. quinquefasciatus., (© 2021. The Author(s).)

Background: Estimates of the geographical distribution of Culex mosquitoes in the Americas have been limited to state and provincial levels in the United States and Canada and based on data from the 1980s. Since these estimates were made, there have been many more documented observations of mosquitoes and new methods have been developed for species distribution modeling. Moreover, mosquito distributions are affected by environmental conditions, which have changed since the 1980s. This calls for updated estimates of these distributions to understand the risk of emerging and re-emerging mosquito-borne diseases., Methods: We used contemporary mosquito data, environmental drivers, and a machine learning ecological niche model to create updated estimates of the geographical range of seven predominant Culex species across North America and South America: Culex erraticus, Culex nigripalpus, Culex pipiens, Culex quinquefasciatus, Culex restuans, Culex salinarius, and Culex tarsalis., Results: We found that Culex mosquito species differ in their geographical range. Each Culex species is sensitive to both natural and human-influenced environmental factors, especially climate and land cover type. Some prefer urban environments instead of rural ones, and some are limited to tropical or humid areas. Many are found throughout the Central Plains of the USA., Conclusions: Our updated contemporary Culex distribution maps may be used to assess mosquito-borne disease risk. It is critical to understand the current geographical distributions of these important disease vectors and the key environmental predictors structuring their distributions not only to assess current risk, but also to understand how they will respond to climate change. Since the environmental predictors structuring the geographical distribution of mosquito species varied, we hypothesize that each species may have a different response to climate change., (© 2021. The Author(s).)

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.

Background: Wolbachia pipientis are endosymbiotic bacteria present in a large proportion of terrestrial arthropods. The species is known to sometimes affect the ability of its host to transmit vector-borne pathogens. Central Sweden is endemic for Sindbis virus (SINV), where it is mainly transmitted by the vector species Culex pipiens and Culex torrentium, with the latter established as the main vector. In this study we investigated the Wolbachia prevalence in these two vector species in a region highly endemic for SINV., Methods: Culex mosquitoes were collected using CDC light traps baited with carbon dioxide over 9 years at 50 collection sites across the River Dalälven floodplains in central Sweden. Mosquito genus was determined morphologically, while a molecular method was used for reliable species determination. The presence of Wolbachia was determined through PCR using general primers targeting the wsp gene and sequencing of selected samples., Results: In total, 676 Cx. pipiens and 293 Cx. torrentium were tested for Wolbachia. The prevalence of Wolbachia in Cx. pipiens was 97% (95% CI 94.8-97.6%), while only 0.7% (95% CI 0.19-2.45%) in Cx. torrentium. The two Cx. torrentium mosquitoes that were infected with Wolbachia carried different types of the bacteria., Conclusions: The main vector of SINV in the investigated endemic region, Cx. torrentium, was seldom infected with Wolbachia, while it was highly prevalent in the secondary vector, Cx. pipiens. The presence of Wolbachia could potentially have an impact on the vector competence of these two species. Furthermore, the detection of Wolbachia in Cx. torrentium could indicate horizontal transmission of the endosymbiont between arthropods of different species., (© 2021. The Author(s).)

Background: Culex (Culex) tritaeniorhynchus is an important vector of Japanese encephalitis virus (JEV) affecting feral pigs, native mammals and humans. The mosquito species is widely distributed throughout Southeast Asia, Africa and Europe, and thought to be absent in Australia., Methods: In February and May, 2020 the Medical Entomology unit of the Northern Territory (NT) Top End Health Service collected Cx. tritaeniorhynchus female specimens (n = 19) from the Darwin and Katherine regions. Specimens were preliminarily identified morphologically as the Vishnui subgroup in subgenus Culex. Molecular identification was performed using cytochrome c oxidase subunit 1 (COI) barcoding, including sequence percentage identity using BLAST and tree-based identification using maximum likelihood analysis in the IQ-TREE software package. Once identified using COI, specimens were reanalysed for diagnostic morphological characters to inform a new taxonomic key to related species from the NT., Results: Sequence percentage analysis of COI revealed that specimens from the NT shared 99.7% nucleotide identity to a haplotype of Cx. tritaeniorhynchus from Dili, Timor-Leste. The phylogenetic analysis showed that the NT specimens formed a monophyletic clade with other Cx. tritaeniorhynchus from Southeast Asia and the Middle East. We provide COI barcodes for most NT species from the Vishnui subgroup to aid future identifications, including the first genetic sequences for Culex (Culex) crinicauda and the undescribed species Culex (Culex) sp. No. 32 of Marks. Useful diagnostic morphological characters were identified and are presented in a taxonomic key to adult females to separate Cx. tritaeniorhynchus from other members of the Vishnui subgroup from the NT., Conclusions: We report the detection of Cx. tritaeniorhynchus in Australia from the Darwin and Katherine regions of the NT. The vector is likely to be already established in northern Australia, given the wide geographical spread throughout the Top End of the NT. The establishment of Cx. tritaeniorhynchus in Australia is a concern to health officials as the species is an important vector of JEV and is now the sixth species from the subgenus Culex capable of vectoring JEV in Australia. We suggest that the species must now be continuously monitored during routine mosquito surveillance programmes to determine its current geographical spread and prevent the potential transmission of exotic JEV throughout Australia., (© 2021. The Author(s).)

While adding taxon names to the Systema Dipterorum database (Evenhuis Pape 2021), one of us (NLE) discovered that Humboldt (1819) had spelled the proposed name of a nominal mosquito species in two ways. He described the species, which was found in swampy places along the Magdalena River near Tenerife, Colombia, as Culex cyanopennis on page 340 and afterwards referred to it as Culex cyanopterus on pages 345 and 349. Both names have the same meaning: cyano- (Gr. kyanos, dark blue), pennis (L. penna, feather, wing) and pteron (Gr. feather, wing). The species was named for the perceived color of the wings: "Alæ cæruleæ, splendore semi-metallico…" (wings blue, a bright semi-metallic). On page 345, Humboldt states, translated from the French: "We have been informed in the Rio de la Magdalena that in Simitì no other Culex than the jejen [je·jén: Sp., gnat, mosquito] was known in the past. You can spend the night there quietly, because the jejen is not a nocturnal insect. Since the year 1801, the big blue-winged mosquito (Culex cyanopterus) has shown itself in such abundance that the poor inhabitants of Simitì do not know how to get a peaceful sleep." Thus, in addition to having the same meaning, the two names are associated with the same locality.

Culex bhutanensis Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex Linnaeus. Based on morphological distinctions and phylogenetic analysis of COI sequences, the new species is a member of the Mimulus Complex. The adults have wing markings similar to those of Cx. mimulus Edwards and Cx. murrelli Lien, but are distinguished by the presence of a line of pale scales or scattered pale scales along the posterior margin of the proximal portion of the costa. The larva is most similar to the larva of Cx. orientalis Edwards from Japan and South Korea, but is distinguished by the form of the comb scales, pecten spines and seta 2-X. The pupa differs from the pupae of other species of the subgroup in having seta 8-C with more branches. The immature stages of the new species were found in rice paddies, stagnant pools and marshes at high altitudes in several districts of Bhutan., (Copyright © 2021. Published by Elsevier B.V.)

Culex modestus mosquitoes are considered potential transmission vectors of West Nile virus and Usutu virus. Their presence has been reported across several European countries, including one larva detected in Belgium in 2018. In this study, mosquitoes were collected in the city of Leuven and surrounding areas in the summers of 2019 and 2020. Species identification was performed based on morphological features and partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The 107 mosquitoes collected in 2019 belonged to eight mosquito species, Culex pipiens (24.3%), Cx. modestus (48.6%), Cx. torrentium (0.9%), Culiseta annulata (0.9%), Culiseta morsitans (0.9%), Aedes sticticus (14.0%), Aedes cinereus (9.3%), and Anopheles plumbeus (0.9%), suggesting the presence of an established Cx. modestus population in Belgium. The collection of Cx. modestus mosquitoes at the same locations in 2020 confirmed their establishment in the region. Haplotype network analysis of the COI sequences for Cx. modestus showed that the Belgian population is rather diverse, suggesting that it may have been established in Belgium for some time. The Belgian Cx. modestus population was most closely related to populations from the United Kingdom and Germany. Characterization of the virome of the collected mosquitoes resulted in the identification of at least 33 eukaryotic viral species. Nine (nearly) complete genomes belonging to 6 viral species were identified, all of which were closely related to known viruses. In conclusion, here, we report the presence of Cx. modestus in the surrounding areas of Leuven, Belgium. As this species is considered to be a vector of several arboviruses, the implementation of vector surveillance programs to monitor this species is recommended. IMPORTANCE Culex modestus mosquitoes are considered to be a potential "bridge" vector, being able to transmit pathogens between birds as well as from birds to mammals, including humans. In Belgium, this mosquito species was considered absent until the finding of one larva in 2018 and subsequent evidence of a large population in 2019 to 2020 described here. We collected mosquitoes in the summers of 2019 and 2020 in the city of Leuven and surrounding areas. The mosquito species was identified by morphological and molecular methods, demonstrating the presence of Cx. modestus in this region. The ability of mosquitoes to transmit pathogens can depend on several factors, one of them being their natural virus composition. Therefore, we identified the mosquito-specific viruses harbored by Belgian mosquitoes. As Cx. modestus is able to transmit viruses such as West Nile virus and Usutu virus, the establishment of this mosquito species may increase the risk of virus transmission in the region. It is thus advisable to implement mosquito surveillance programs to monitor this species., (Copyright © 2021 Wang et al.)

Despite the importance of Culex species as major vectors of Rift Valley fever virus, West Nile virus and the microfilariae that cause lymphatic filariasis, information on these mosquitoes in Sudan is limited to works published 65 years ago in the former Anglo-Egyptian Sudan, where some species were only recorded from areas of the territory now known as South Sudan. In this paper, we provide updated information on Culex mosquitoes collected indoors during surveillance studies conducted along the Nile River in central and northern areas of Sudan between 2012 and 2019. Of 3,411 female mosquitoes collected in Khartoum and northern states along the river, 2,560 (75%) were specimens of Culex belonging to 12 species: Cx. (Culex) antennatus (Becker, 1903), Cx. (Cux.) laticinctus Edwards, 1913, Cx. (Cux.) neavei Theobald, 1906, Cx. (Cux.) pipiens Linnaeus, 1758, Cx. (Cux.) perexiguus Theobald, 1903, Cx. (Cux.) poicilipes (Theobald, 1903), Cx. (Cux.) quinquefasciatus Say, 1823, Cx. (Cux.) simpsoni Theobald, 1905, Cx. (Cux.) sinaiticus Kirkpatrick, 1925, Cx. (Cux.) theileri Theobald, 1903, Cx. (Cux.) tritaeniorhynchus Giles, 1901 and Cx. (Culiciomyia) macfiei Edwards, 1923. This is the first record for Cx. tritaeniorhynchus and Cx. macfiei in central Sudan. The relative abundance of each species varied in different areas and seasons, but Cx. antennatus and Cx. quinquefasciatus were the most abundant indoor resting species. We provide an updated dichotomous key for the identification of the adults of Culex mosquitoes known to occur in the Republic of the Sudan.

Background: Chikungunya virus is an alphavirus, primarily transmitted by Aedes aegypti and Ae. albopictus. In late 2017-2018, an outbreak of chikungunya occurred in Mombasa county, Kenya, and investigations were conducted to establish associated entomological risk factors., Methods: Homes were stratified and water-filled containers inspected for immature Ae. aegypti, and larval indices were calculated. Adult mosquitoes were collected in the same homesteads using BG-Sentinel and CDC light traps and screened for chikungunya virus. Experiments were also conducted to determine the ability of Culex quinquefasciatus to transmit chikungunya virus., Results: One hundred thirty-one houses and 1637 containers were inspected; 48 and 128 of them, respectively, were positive for immature Ae. aegypti, with the house index (36.60), container index (7.82) and Breteau index (97.71) recorded. Jerry cans (n = 1232; 72.26%) and clay pots (n = 2; 0.12%) were the most and least inspected containers, respectively, while drums, the second most commonly sampled (n = 249; 15.21%), were highly positive (65.63%) and productive (60%). Tires and jerry cans demonstrated the highest and lowest breeding preference ratios, 11.36 and 0.2, respectively. Over 6900 adult mosquitoes were collected and identified into 15 species comprising Cx. quinquefasciatus (n = 4492; 65.04%), Aedes vittatus (n = 1137; 16.46%) and Ae. aegypti (n = 911; 13.19%) and 2 species groups. Simpson's dominance and Shannon-Wiener diversity indices of 0.4388 and 1.1942 were recorded, respectively. Chikungunya virus was isolated from pools of Ae. aegypti (1) and Cx. quinquefasciatus (4), two of which were males. Minimum infection rates of 3.0 and 0.8 were observed for female Ae. aegypti and Cx. quinquefasciatus, respectively. Between 25 and 31.3% of exposed mosquitoes became infected with CHIKV 7, 14 and 21 days post-exposure. For the experimentally infected Cx. quinquefasciatus mosquitoes, between 13 and 40% had the virus disseminated, with 100% transmission being observed among those with disseminated infection., Conclusions: These results demonstrated high risk of chikungunya transmission for residents in the sampled areas of Mombasa. Transmission data confirmed the probable role played by Cx. quinquefasciatus in the outbreak while the role of Ae. vittatus in the transmission of chikungunya virus remains unknown.

The taxonomy of Culex pipiens complex of mosquitoes is still debated, but in North America it is generally regarded to include Culex pipiens pipiens, Culex pipiens molestus, and Culex quinquefasciatus (or Culex pipiens quinquefasciatus). Although these mosquitoes have very similar morphometry, they each have unique life strategies specifically adapted to their ecological niche. Differences include the capability for overwintering diapause, bloodmeal preference, mating behaviors, and reliance on blood meals to produce eggs. Here, we used RNA-seq transcriptome analysis to investigate the differential gene expression and nucleotide polymorphisms that may link to the divergent traits specifically between Cx. pipiens pipiens and Cx. pipiens molestus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We conducted a nationwide survey of mosquito distribution in Malawi from November 2011 to April 2012, and from July to September 2012. Using dried specimens of mosquito adults collected during the survey, we analyzed their cytochrome c oxidase subunit I (COI) gene sequences, prepared specimens, and registered the genetic information (658 bp) of 144 individuals belonging to 51 species of 10 genera in GenBank. Using the obtained genetic information, we analyzed the degree of intraspecific variation and investigated the various species from morphological and genetic perspectives. Moreover, we conducted phylogenetic analysis of the medically important species distributed from Africa to Asia and explored their geographical differentiation. Results showed that individuals morphologically classified as Culex univittatus complex included a individual of Cx. perexiguus which, to date, have not been reported in southern Africa. Furthermore, Mansonia uniformis, distributed in Africa and Asia, was revealed to belong to genetically distinct populations, with observed morphological differences of the samples suggesting that they are separate species. The results of genetic analysis further suggested that Cx. ethiopicus is not a synonym of Cx. bitaeniorhynchus, but that it is an independent species; although, in this study, the only definite morphological difference observed was in the shape of the wing scales. Further morphological and genetic investigation of individuals of these species, including larvae, is highly recommended., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Several mosquito-borne viruses (mobovirus) cause infections in Canada. Ecological data on mosquito species and host range in Canada remains elusive. The main aim of the current study is to determine the host range and molecular systematics of mosquito species in Canada. Mosquitoes were collected using BG-Sentinel traps and aspirators at 10 trapping sites in Canada during 2018 and 2019. Mosquitoes collected were identified via morphology and molecular techniques. Mosquito sequences were aligned by MUSCLE algorithm and evolutionary systematics were drawn using MEGA and SDT software. Moreover, the source of blood meals was identified using a DNA barcoding technique. A total of 5,708 female mosquitoes over 34 different taxa were collected. DNA barcodes and evolutionary tree analysis confirmed the identification of mosquito species in Canada. Of the total collected samples, 201 specimens were blood-fed female mosquitoes in 20 different taxa. Four mosquito species represented about half (51.47%) of all collected blood-fed specimens: Aede cinereus (39 specimens, 19.11%), Aedes triseriatus (23, 11.27%), Culex pipiens (22, 10.78%), and Anopheles punctipennis (21, 10.29%). The most common blood meal sources were humans (49 mosquito specimens, 24% of all blood-fed mosquito specimen), pigs (44, 21.5%), American red squirrels (28, 13.7%), white-tailed deers (28, 13.7%), and American crows (16, 7.8%). Here, we present the first analysis of the host-feeding preference of different mosquito species in Canada via molecular techniques. Our results on mosquito distribution and behavior will aid in the development of effective mitigation and control strategies to prevent or reduce human/animal health issues in regards to moboviruses., (Copyright © 2020. Published by Elsevier B.V.)

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico., (Copyright © 2020. Published by Elsevier B.V.)

Background: The mosquito species Culex pipiens is a known vector of several pathogens and occurs in two distinct bioforms, pipiens and molestus . The bioform molestus thrives in urban environments where there are below-ground habitats; it can mate in confined spaces and feed on mammals as well as birds. In contrast, the bioform pipiens is found above ground, is thought to require more space for mating, and mainly feeds on birds. The pipiens bioform is present in large parts of Sweden but the molestus bioform has previously only been found in major cities. Materials and Methods: People experiencing mosquito nuisance in southern Sweden submitted mosquito samples as part of a citizen science project, and these samples were analyzed to determine the geographical distribution of the molestus bioform of Cx. pipiens . Mosquito specimens were identified to the species level by DNA barcoding of the cytochrome C oxidase subunit I (COI) gene, and the bioforms were determined through the CQ11 microsatellite marker. Results: Culex pipiens f molestus was observed to be spread across large parts of Gothenburg as well as in the suburbs. This bioform was found both in urban and rural areas at several sites across southern Sweden. In one site, hybrids between the two bioforms were found. Conclusions: The detection of Cx. pipiens f molestus in several rural areas was surprising, indicating that it may be more widely spread than urban areas alone, where it has been previously reported.

As vectors of malaria, dengue, zika, and yellow fever, mosquitoes are considered one of the more severe worldwide health hazards. Widespread surveillance of mosquitoes is essential for understanding their complex ecology and behaviour, and also for predicting and formulating effective control strategies against mosquito-borne diseases. One technique involves using bioacoustics to automatically identify different species from their wing-beat sounds during flight. In this dataset, we collect sounds of three species of mosquitoes: Aedes Aegypti, Culex Quinquefasciatus & Pipiens, and Culiseta. These species were collected and reproduced in the laboratory of the Natural History Museum of Funchal, in Portugal, by entomologists trained to recognize and classify mosquitoes. For collecting the samples, we used a microcontroller and a mobile phone. The dataset presents audio samples collected with different sampling rates, where 34 audio features characterize each sound file, making it is possible to observe how mosquito populations vary heterogeneously. This dataset provides the basis for feature extraction and classification of flapping-wing flight sounds that could be used to identify different species.

Mosquitoes (Diptera: Culicidae) in the Culex pipiens complex play a key role in the transmission and therefore epidemiology of a number of human and animal pathogens globally. These mosquitoes, and sympatric species of the genus Culex Linnaeus that are not within the Cx. pipiens complex, are often considered 'impossible' to distinguish by morphology in the adult female stage. In the United States, this is particularly true for Culex pipiens s.l. and Culex restuans Theobald, both of which are competent vectors of West Nile virus, but likely play different roles in the transmission cycle. Therefore, we undertook an in-depth morphological evaluation of matched larval exuviae and adult specimens that revealed five useful morphological characters that are informative to distinguish Cx. pipiens s.l. from Cx. restuans in the adult stage. Herein, we provide a comprehensive review of the literature on these species of interest, and four additional, morphologically similar, Culex species, and a proposed key to adult female specimens., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak., Methods: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis., Results: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo., Conclusions: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

Background: Mosquitoes are vectors of many tropical diseases. Understanding the ecology of local mosquito vectors, such as species composition, distributions, population dynamics, and species diversity is important for designing the optimal strategy to control the mosquito-borne diseases., Methods: Entomological surveillance of adult mosquitoes was conducted in five sites representing different ecological settings across Hainan Island from January to December of 2018 using BG Sentinel (BGS) traps and Centers for Disease Prevention and Control (CDC) light traps. In each site, we selected three areas representing urban, suburban and rural settings. Eighteen trap-days were sampled in each setting at each site, and CDC light traps and BGS traps were setup simultaneously. Mosquito species composition, distribution, population dynamics, and species diversity were analyzed. Mosquito densities were compared between different study sites and between different settings., Results: Nine species of mosquitoes belonging to four genera were identified. Culex quinquefasciatus (80.8%), Armigeres subalbatus (13.0%) and Anopheles sinensis (3.1%) were the top three species collected by CDC light traps; Cx. quinquefasciatus (91.9%), Ae. albopictus (5.1%), and Ar. subalbatus (2.8%) were the top three species collected by BGS traps. Predominant species varied among study sites. The population dynamics of Ae. albopictus, An. sinensis and Cx. quinquefasciatus showed clear seasonal variation regardless of study sites with a varied peak season for different species. Mosquito abundance of all species showed significant differences among different study sites and among urban, suburban and rural areas. Danzhou had the highest mosquito biodiversity, with an α, β, and Gini-Simpson biodiversity index of 8, 1.13 and 0.42, respectively. BGS traps captured Aedes mosquito at a higher efficiency than CDC light traps, whereas CDC light traps captured significantly more Anopheles and Armigeres mosquitoes than BGS traps., Conclusions: Mosquitoes were abundant on Hainan Island with clear seasonality and spatial heterogeneity. Population density, species composition, distribution, and species diversity were strongly affected by the natural environment. Different tools are required for the surveillance of different mosquito species.

Before the background of increasingly frequent outbreaks and cases of mosquito-borne diseases in various European countries, Germany recently realised the necessity of updating decade-old data on the occurrence and spatiotemporal distribution of culicid species. Starting in 2011, a mosquito monitoring programme was therefore launched with adult and immature mosquito stages being collected at numerous sites all over Germany both actively by trapping, netting, aspirating and dipping, and passively by the citizen science project 'Mueckenatlas'. Until the end of 2019, about 516,000 mosquito specimens were analysed, with 52 (probably 53) species belonging to seven genera found, including several species not reported for decades due to being extremely rare (Aedes refiki, Anopheles algeriensis, Culex martinii) or local (Culiseta alaskaensis, Cs. glaphyroptera, Cs. ochroptera). In addition to 43 (probably 44 including Cs. subochrea) out of 46 species previously described for Germany, nine species were collected that had never been documented before. These consisted of five species recently established (Ae. albopictus, Ae. japonicus, Ae. koreicus, An. petragnani, Cs. longiareolata), three species probably introduced on one single occasion only and not established (Ae. aegypti, Ae. berlandi, Ae. pulcritarsis), and a newly described cryptic species of the Anopheles maculipennis complex (An. daciae) that had probably always been present but not been differentiated from its siblings. Two species formerly listed for Germany could not be documented (Ae. cyprius, Ae. nigrinus), while presence is likely for another species (Cs. subochrea), which could not be demonstrated in the monitoring programme as it can neither morphologically nor genetically be reliably distinguished from a closely related species (Cs. annulata) in the female sex. While Cs. annulata males were collected in the present programme, this was not the case with Cs. subochrea. In summary, although some species regarded endemic could not be found during the last 9 years, the number of culicid species that must be considered firmly established in Germany has increased to 51 (assuming Cs. subochrea and Ae. nigrinus are still present) due to several newly emerged ones but also to one species (Ae. cyprius) that must be considered extinct after almost a century without documentation. Most likely, introduction and establishment of the new species are a consequence of globalisation and climate warming, as three of them are native to Asia (Ae. albopictus, Ae. japonicus, Ae. koreicus) and three (Ae. albopictus, An. petragnani, Cs. longiareolata) are relatively thermophilic. Another thermophilic species, Uranotaenia unguiculata, which had been described for southwestern Germany in 1994 and had since been found only at the very site of its first detection, was recently documented at additional localities in the northeastern part of the country. As several mosquito species found in Germany are serious pests or potential vectors of disease agents and should be kept under permanent observation or even be controlled immediately on emergence, the German mosquito monitoring programme has recently been institutionalised and perpetuated.

Background: Twenty-seven villages were selected in southwest Burkina Faso to implement new vector control strategies in addition to long lasting insecticidal nets (LLINs) through a Randomized Controlled Trial (RCT). We conducted entomological surveys in the villages during the dry cold season (January 2017), dry hot season (March 2017) and rainy season (June 2017) to describe malaria vectors bionomics, insecticide resistance and transmission prior to this trial., Methods: We carried out hourly catches (from 17:00 to 09:00) inside and outside 4 houses in each village using the Human Landing Catch technique. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were identified using molecular techniques as well as detection of Plasmodium falciparum infection and insecticide resistance target-site mutations., Results: Eight Anopheles species were detected in the area. Anopheles funestus s.s was the main vector during the dry cold season. It was replaced by Anopheles coluzzii during the dry hot season whereas An. coluzzii and An. gambiae s.s. were the dominant species during the rainy season. Species composition of the Anopheles population varied significantly among seasons. All insecticide resistance mechanisms (kdr-w, kdr-e and ace-1 target site mutations) investigated were found in each members of the An. gambiae complex but at different frequencies. We observed early and late biting phenotypes in the main malaria vector species. Entomological inoculation rates were 2.61, 2.67 and 11.25 infected bites per human per month during dry cold season, dry hot season and rainy season, respectively., Conclusion: The entomological indicators of malaria transmission were high despite the universal coverage with LLINs. We detected early and late biting phenotypes in the main malaria vector species as well as physiological insecticide resistance mechanisms. These data will be used to evaluate the impact of complementary tools to LLINs in an upcoming RCT., Competing Interests: The authors have declared that no competing interests exist.

Background: Mosquito vectors cause a significant human public health burden through the transmission of pathogens. Due to the expansion of international travel and trade, the dispersal of these mosquito vectors and the pathogens they carry is on the rise. Entomological surveillance is therefore required which relies on accurate mosquito species identification. This study aimed to optimize the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for mosquito identification., Methods: Aedes aegypti of the Bora-Bora strain and 11 field-sampled mosquito species were used in this study. Analyses were performed to study the impact of the trapping duration on mosquito identification with MALDI-TOF MS. The best preservation methods to use for short, medium and long-term preservation before MALDI-TOF MS analysis were also assessed. In addition, the number of specimens per species required for MALDI-TOF MS database creation was determined. The first MALDI-TOF database of New Caledonian mosquitoes was assembled and the optimal threshold for mosquito species identification according to the sensitivity and specificity of this technique was determined., Results: This study showed that the identification scores decreased as the trapping duration increased. High identification scores were obtained for mosquitoes preserved on silica gel and cotton at room temperature and those frozen at - 20 °C, even after two months of preservation. In addition, the results showed that the scores increased according to the number of main spectrum patterns (MSPs) used until they reached a plateau at 5 MSPs for Ae. aegypti. Mosquitoes (n = 67) belonging to 11 species were used to create the MALDI-TOF reference database. During blind test analysis, 96% of mosquitoes tested (n = 224) were correctly identified. Finally, based on MALDI-TOF MS sensitivity and specificity, the threshold value of 1.8 was retained for a secure identification score., Conclusions: MALDI-TOF MS allows accurate species identification with high sensitivity and specificity and is a promising tool in public health for mosquito vector surveillance.

Most data on species associations and vector potential of mosquitoes in relation to arboviral infections in South Africa date back from the 1940s to late 1990s. Contextual information crucial for disease risk management and control, such as the sampling effort, diversity, abundance, and distribution of mosquitoes in large parts of South Africa still remains limited. Adult mosquitoes were collected routinely from two horse farms in Gauteng Province; two wildlife reserves in Limpopo Province, at Orpen Gate in Kruger National Park (KNP) and Mnisi Area in Mpumalanga Province between 2014-2017, using carbon dioxide-baited light and tent traps. Mosquito diversity and richness are greater in untransformed natural and mixed rural settings. In untransformed wilderness areas, the most dominant species were Culex poicilipes, Anopheles coustani, and Aedes mcintoshi, while in mixed rural settings such as the Mnisi area, the two most abundant species were Cx. poicilipes and Mansonia uniformis. However, in peri-urban areas, Cx. theileri, Cx. univittatus, and Cx. pipiens sensu lato were the most dominant. Aedes aegypti, Ae. mcintoshi, Ae. metallicus, Ae. vittatus, Cx. pipiens s.l., Cx. theileri, and Cx. univittatus had the widest geographical distribution in northern South Africa. Also collected were Anopheles arabiensis and An. vaneedeni, both known malaria vectors in South Africa. Arbovirus surveillance and vector control programs should be augmented in mixed rural and peri-urban areas where the risk for mosquito-borne disease transmission to humans and domestic stock is greater., (© 2020 The Society for Vector Ecology.)

Background: Despite the importance of some species of Culex (Melanoconion) (Diptera: Culicidae) as vectors of several arboviruses that cause diseases in humans and other animals, there are few taxonomic studies focusing on species of the subgenus, especially providing morphological keys for species identification., Results: Thirteen species of the Atratus Group of Culex (Melanoconion) were reviewed, five new species are described, and two taxonomic changes are proposed: Cx. (Mel.) exedrus Root, 1927 and Cx. (Mel.) loturus Dyar, 1925 are resurrected from synonymy with Cx. (Mel.) dunni Dyar, 1918 and Cx. (Mel.) zeteki Dyar, 1918, respectively. The Atratus Group now includes fourteen species: Cx. (Mel.) atratus Theobald, 1901; Cx. (Mel.) caribeanus Galindo & Blanton, 1954; Cx. (Mel.) columnaris Sá & Hutchings n. sp.; Cx. (Mel.) commevynensis Bonne-Wepster & Bonne, 1919; Cx. (Mel.) comptus Sá & Sallum n. sp.; Cx. (Mel.) dunni; Cx. (Mel.) ensiformis Bonne-Wepster & Bonne, 1919; Cx. (Mel.) exedrus; Cx. (Mel.) longisetosus Sá & Sallum n. sp.; Cx. (Mel.) longistylus Sá & Sallum n. sp.; Cx. (Mel.) loturus; Cx. (Mel.) spinifer Sá & Sallum n. sp.; Cx. (Mel.) trigeminatus Clastrier, 1970; and Cx. (Mel.) zeteki. Keys, descriptions and illustrations for the identification of the male, female, pupal and fourth-instar larval stages of each species are provided. The treatment of each species includes a complete synonymy, descriptions of available life stages, a taxonomic discussion, updated bionomics and geographical distribution, and a list of material examined., Conclusions: The taxonomy of the Atratus Group of Culex (Melanoconion) is updated, including descriptions of five new species. The number of valid species is greater than the number recognized in the previous taxonomic study of the group, increasing from seven to 14 species. Distributional and bionomical data are updated. Morphology-based identification keys for females, males, fourth-instar larvae and pupae provided in this study will facilitate species identification.

Understanding the population structure and mechanisms of taxa diversification is important for organisms responsible for the transmission of human diseases. Two vectors of West Nile virus, Culex pipiens pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The goal of this study is to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations but not in Cx. p. molestus populations. Comparison of mitochondrial genomes among the specimens showed a paraphyletic origin of the major haplogroups between the subspecies but a monophyletic structure between the continents. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation.

Japanese encephalitis (JE) remains a public health concern in several countries, and the Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus-ISV interactions; however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C . pseudovishnui , which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C . inatomii . A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae , Rhabdoviridae , Totiviridae , and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure (e.g., virome composition, diversity, and abundance) as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors' knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.

Image-based automatic classification of vector mosquitoes has been investigated for decades for its practical applications such as early detection of potential mosquitoes-borne diseases. However, the classification accuracy of previous approaches has never been close to human experts' and often images of mosquitoes with certain postures and body parts, such as flatbed wings, are required to achieve good classification performance. Deep convolutional neural networks (DCNNs) are state-of-the-art approach to extracting visual features and classifying objects, and, hence, there exists great interest in applying DCNNs for the classification of vector mosquitoes from easy-to-acquire images. In this study, we investigated the capability of state-of-the-art deep learning models in classifying mosquito species having high inter-species similarity and intra-species variations. Since no off-the-shelf dataset was available capturing the variability of typical field-captured mosquitoes, we constructed a dataset with about 3,600 images of 8 mosquito species with various postures and deformation conditions. To further address data scarcity problems, we investigated the feasibility of transferring general features learned from generic dataset to the mosquito classification. Our result demonstrated that more than 97% classification accuracy can be achieved by fine-tuning general features if proper data augmentation techniques are applied together. Further, we analyzed how this high classification accuracy can be achieved by visualizing discriminative regions used by deep learning models. Our results showed that deep learning models exploit morphological features similar to those used by human experts.

Background: Within the Culex pipiens mosquito complex, there are six contemporarily recognized taxa: Cx. quinquefasciatus, Cx. pipiens f. pipiens, Cx. pipiens f. molestus, Cx. pipiens pallens, Cx. australicus and Cx. globocoxitus. Many phylogenetic aspects within this complex have eluded resolution, such as the relationship of the two Australian endemic taxa to the other four members, as well as the evolutionary origins and taxonomic status of Cx. pipiens pallens and Cx. pipiens f. molestus. Ultimately, insights into lineage relationships within the complex will facilitate a better understanding of differential disease transmission by these mosquitoes. To this end, we have combined publicly available data with our own sequencing efforts to examine these questions., Results: We found that the two Australian endemic complex members, Cx. australicus and Cx. globocoxitus, comprise a monophyletic group, are genetically distinct, and are most closely related to the cosmopolitan Cx. quinquefasciatus. Our results also show that Cx. pipiens pallens is genetically distinct, but may have arisen from past hybridization. Lastly, we observed complicated patterns of genetic differentiation within and between Cx. pipiens f. pipiens and Cx. pipiens f. molestus., Conclusions: Two Australian endemic Culex taxa, Cx. australicus and Cx. globocoxitus, belong within the Cx. pipiens complex, but have a relatively older evolutionary origin. They likely diverged from Cx. quinquefasciatus after its colonization of Australia. The taxon Cx. pipiens pallens is a distinct evolutionary entity that likely arose from past hybridization between Cx. quinquefasciatus and Cx. pipiens f. pipiens/Cx. pipiens f. molestus. Our results do not suggest it derives from ongoing hybridization. Finally, genetic differentiation within the Cx. pipiens f. pipiens and Cx. pipiens f. molestus samples suggests that they collectively form two separate geographic clades, one in North America and one in Europe and the Mediterranean. This may indicate that the Cx. pipiens f. molestus form has two distinct origins, arising from Cx. pipiens f. pipiens in each region. However, ongoing genetic exchange within and between these taxa have obscured their evolutionary histories, and could also explain the absence of monophyly among our samples. Overall, this work suggests many avenues that warrant further investigation.

A gynandromorph of Culex sitiens Wiedemann (Diptera: Culicidae) was attracted to a human during a mosquito surveillance programme conducted in Kuala Lipis, Pahang, Malaysia on July 20, 2019. Gynandromorphism was observed in antennae, maxillary palps, legs and wings of the specimen, with distinct male characters on the left and female characters on the right, though the left maxillary palp is slightly shorter than the proboscis of a typical male. The abdomen, however, displays well-developed male genitalia. This study represents the first report of oblique gynandromorphism in Cx. sitiens, one of the vectors of Japanese encephalitis in Asia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background & Objectives: Insecticides are the most important means of controlling pests in Iran especially for Culex pipiens complex species. The rational use of insecticides largely depends on understanding the susceptibility levels of these species. The study was designed to determine the susceptibility levels of Cx. pipiens complex (field and insectary strains) to various insecticides in the city of Tehran., Methods: The mortality rates of the field strain of Cx. pipiens complex after different exposure times to DDT (4%), bendiocarb (0.1%), propoxur (0.1%), malathion (5%), fenitrothion (1.0%), permethrin (0.75%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%), etofenprox (0.5%), and cyfluthrin (0.15%) were determined. The mortality rates at the lethal time 50% (LT 50 ) and lethal time 90% (LT 90 ) values were calculated by plotting the regression line using Microsoft Office Excel software., Results: The mortality rates of the Cx. pipiens complex after 1 h exposure to the diagnostic doses of DDT (4%), bendiocarb (0.1%), propoxur (0.1%), malathion (5%), fenitrothion (1.0%), permethrin (0.75%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%), etofenprox (0.5%), and cyfluthrin (0.15%) were 12, 58, 54, 82, 54, 34, 49, 40, 17, and 44%, respectively. According to the WHO classification of susceptibility levels, both field and insectary strains of Cx. pipiens complex in Tehran were resistant to these insecticides., Interpretation & Conclusion: The results of this study showed that field Cx. pipiens complex is resistant to all the groups of insecticides used., Competing Interests: None

Introduction: This study aimed to assess the occurrence of gonotrophic discordance in females of Culex quinquefasciatus in São Paulo, Brazil., Methods: Resting females were collected monthly for 8 months. Females of Cx. quinquefasciatus were identified, and their midgut and ovaries were dissected., Results: Two hundred females were dissected, out of which, 27.5% were nulliparous and 57% were parous. Most females had no blood in the midgut, but gonotrophic discordance was found in 21% females., Conclusions: Females of Cx. quinquefasciatus showed a high parity rate and gonotrophic discordance, which could favor the vector capacity of this species.

The northern house mosquito (Culex pipiens pipiens L.) is a vector of several important pathogens and comprises two epidemiologically distinct ecotypes (molestus Forskål and pipiens). The delimitation of its ecotypes is a crucial, yet controversial step in vector surveillance due to varying diagnostic values of different characters. Therefore, we reviewed the success of a diagnostic assay based on the mitochondrial cytochrome c oxidase subunit I locus (COI) by analyzing previously published sequences of molestus and pipiens sampled in different geographical areas. Next, by genotyping individuals from Northern Serbia at this locus, we additionally assessed whether genetic structure of urban and rural Cx. p. pipiens ecotypes corresponded to the admixture pattern. Finally, to account for the different susceptibility of genetic markers to introgression, we also analyzed genetic structuring based on the ribosomal internal transcribed spacer 2 (ITS2). No latitude-dependent differentiation of Cx. p. pipiens ecotypes was found at a global level, with the COI assay further failing to accurately identify molestus and pipiens ecotypes. Likewise, both individual- (BAPS) and population-based (analysis of molecular variance and FST estimates) methods showed no significant urban/rural genetic differentiation in Serbia, indicating unhindered gene flow between different Cx. p. pipiens habitat types. The findings challenge the previous instances of Cx. p. pipiens ecotype identification, while also spotlighting the vectorial capacity of their hybrid offspring.