Automated classification of mixed populations of Aedes aegypti and Culex quinquefasciatus mosquitoes under field conditions.

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.

Mosquito dynamics and their drivers in peri-urban Antananarivo, Madagascar: insights from a longitudinal multi-host single-site survey.

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.

Taxonomic history, biology and ecology of Culex (Microculex) (Diptera: Culicidae).

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.

Host attraction and host feeding patterns indicate generalist feeding of Culex pipiens s.s. and Cx. torrentium.

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.

Application of circular statistics in temporal distribution of adult mosquitoes in Qingdao, Shandong Province, China, 2021-2023.

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.

Resolution of cryptic mosquito species through wing morphometrics.

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.

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.

Description of New Morphological Variation of Culex (Culex) coronator Dyar and Knab, 1906 and First Report of Culex (Carrollia) bonnei Dyar, 1921 Found in the Central Region of Peru.

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.

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.

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.

Application of computer vision and deep learning models to automatically classify medically important mosquitoes in North Borneo, Malaysia.

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.

Diversity, distribution and relative abundance of the mosquito fauna (Diptera: Culicidae) of Malakand and Dir Lower, Pakistan / Diversidade, distribuição e abundância relativa da fauna de mosquitos (Diptera: Culicidae) de Malakand e Dir Lower, Paquistão

The present study was conducted to evaluate the diversity, distribution (C) and relative abundance (RA) of the mosquito fauna (Diptera: Culicidae) of Malakand and Dir Lower, Pakistan. Collection of specimens (n = 1087) was made during September 2018 to July 2019 at six different habitats including freshwater bodies, rice fields, animal sheds, indoors, drains and sewage waters. Specimens were collected through light traps, pyrethrum spray, aspirators and nets and subsequently killed, preserved and then arranged in entomological boxes for identification. Three genera were identified namely Culex, Anopheles and Aedes. A total of fourteen species were identified namely: Cx. quinquefasciatus (Say, 1823), An. stephensi (Liston, 1901), Cx. tritaeniorhynchus (Giles, 1901), Ae. vittatus (Bigot, 1861), An. maculatus (Theobald, 1901), An. fluviatilis (James, 1902), Cx. vishnui (Theobald, 1901), Ae. aegypti (Linnaeus, 1762) An. subpictus (Grassi, 1899), An. dthali (Patton, 1905), An. culicifascies (Giles, 1901), An. pallidus (Theobald, 1901), Ae. albopictus (Skuse, 1894) and An. annularis (van der Wulp, 1884). Cx. quinquefasciatus was found constantly distributed in the study area with RA = 16.5% and C = 100%. An. annularis was found as a satellite species, sporadically distributed in the study area having RA = 0.9% and C = 17%. Diversity indices of mosquitoes in the studied habitats were found as, Shannon-Wiener Index (2.415), Simpson Index (9.919), Fisher's Index (2.269) and Margalef's Index (1.859). A statistically significant difference was recorded in mosquito diversity in the six habitats (Kruskal-Wallis, chi-squared, H = 17.5, df = 5, P = 0.003 at α = 0.05). The present study encompasses [...].

O presente estudo foi conduzido para avaliar a diversidade, distribuição (C) e abundância relativa (RA) da fauna de mosquitos (Diptera: Culicidae) de Malakand e Dir Lower, Paquistão. A coleta de espécimes (n = 1087) foi feita durante o período de setembro de 2018 a julho de 2019 em seis habitats diferentes, incluindo corpos d’água, campos de arroz, galpões de animais, ambientes internos, ralos e águas residuais. Os espécimes foram coletados por meio de armadilhas luminosas, spray de piretro, aspiradores e redes e posteriormente mortos, preservados e depois dispostos em caixas entomológicas para identificação. Três gêneros foram identificados, nomeadamente Culex, Anopheles e Aedes. Um total de 14 espécies foi identificado, a saber: Cx. quinquefasciatus (Say, 1823), An. stephensi (Liston, 1901), Cx. tritaeniorhynchus (Giles, 1901), Ae. vittatus (Bigot, 1861), An. maculatus (Theobald, 1901), An. fluviatilis (James, 1902), Cx. vishnui (Theobald, 1901), Ae. aegypti (Linnaeus, 1762), An. subpictus (Grassi, 1899), An. dthali (Patton, 1905), An. culicifascies (Giles, 1901), An. pallidus (Theobald, 1901), Ae. albopictus (Skuse, 1894) e An. annularis (Van der Wulp, 1884). Cx. quinquefasciatus foi encontrado constantemente distribuído na área de estudo com AR = 16,5% e C = 100%. A. annularis foi encontrada como espécie satélite, distribuída esporadicamente na área de estudo com RA = 0,9% e C = 17%. Os índices de diversidade de mosquitos nos habitats estudados foram encontrados como índice de Shannon-Wiener (2,415), índice de Simpson (9,919), índice de Fisher (2,269) e índice de Margalef (1,859). Uma diferença estatisticamente significativa foi registrada na diversidade de mosquitos nos seis habitats (Kruskal-Wallis, qui-quadrado, H = 17,5, df = 5, P = 0,003 em α = 0,05). O presente estudo abrange a fauna de mosquitos de Malakand, Paquistão, com respeito à diversidade, abundância relativa e distribuição em [...].

Screening of olfactory genes related to blood-feeding behaviors in Culex pipiens quinquefasciatus and Culex pipiens molestus by transcriptome analysis.

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.

Deltamethrin interacts with Culex quinquefasciatus odorant-binding protein: a novel potential resistance mechanism.

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.

Spatial patterns of West Nile virus distribution in the Volgograd region of Russia, a territory with long-existing foci.

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.

Insecticide resistance to insect growth regulators, avermectins, spinosyns and diamides in Culex quinquefasciatus in Saudi Arabia.

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.

Updated distribution maps of predominant Culex mosquitoes across the Americas.

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.

The Effect of Fluctuating Incubation Temperatures on West Nile Virus Infection in Culex Mosquitoes.

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.

Detection of the Japanese encephalitis vector mosquito Culex tritaeniorhynchus in Australia using molecular diagnostics and morphology.

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.