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.

Wolbachia prevalence in the vector species Culex pipiens and Culex torrentium in a Sindbis virus-endemic region of Sweden.

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.

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.

A case of two spellings for Humboldt's blue-winged mosquito (Diptera: Culicidae).

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 (Culex) longitubus, A New Species of the Mimeticus Subgroup (Diptera: Culicidae) From Bhutan.

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.

Culex/ mosquitoes (Diptera: Culicidae) recorded along the Nile River in central and northern Sudan, with a key for the identification of all species of the genus known to occur in the country.

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.

Establishment of Culex modestus in Belgium and a Glance into the Virome of Belgian Mosquito Species.

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.IMPORTANCECulex 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.

Probable contribution of Culex quinquefasciatus mosquitoes to the circulation of chikungunya virus during an outbreak in Mombasa County, Kenya, 2017-2018.

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.

Culex bhutanensis, a new species of the Mimeticus Subgroup of the nominotypical subgenus of the genus Culex Linnaeus (Diptera: Culicidae) from Bhutan.

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.

Identification of mosquitoes (Diptera: Culicidae) from Mexico State, Mexico using morphology and COI DNA barcoding.

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.

Transcript Assembly and Quantification by RNA-Seq Reveals Significant Differences in Gene Expression and Genetic Variants in Mosquitoes of the Culex pipiens (Diptera: Culicidae) Complex.

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.

DNA barcoding of mosquitoes collected through a nationwide survey in 2011 and 2012 in Malawi, Southeast Africa.

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.

Modeling host-feeding preference and molecular systematics of mosquitoes in different ecological niches in Canada.

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.

An Evaluation of Characters for the Separation of Two Culex Species (Diptera: Culicidae) Based on Material From the Upper Midwest.

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.