Molecular Identification of Species Belonging to Culex vishnui Subgroup (Diptera: Culicidae), Vectors of Japanese Encephalitis Virus, in Taiwan.

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.

Species Composition and Feeding Behaviors of Vector Mosquitoes of Avian Infectious Diseases at a Wild Bird Rehabilitation Facility in Japan.

Although wild bird rehabilitation facilities are important for the conservation of wild species, individuals may be kept within the facilities for long periods, consequently posing a risk for the bird to be infected with pathogens to which they are not naturally exposed. In turn, novel pathogens may be introduced through rescued migratory species. Avian malaria and West Nile fever are important avian diseases transmitted by mosquitoes. To understand the transmission dynamics of such diseases at rehabilitation facilities, the ecology of vector mosquitoes, including species composition, seasonality, and feeding behaviors, were explored. Mosquitoes were collected at a wild bird rehabilitation facility and wildlife sanctuary in Japan from 2019 to 2020 using mouth aspirators, sweep nets, and light traps. A total of 2,819 mosquitoes of 6 species were captured, all of which are potential vectors of avian diseases. Culex pipiens pallens and Cx. pipiens form molestus were the dominant species (82.9% of all collected mosquitoes). Density and seasonality differed between sampling locations, presumably because of differences in mosquito behaviors including feeding preferences and responses to climatic factors. Blood-fed Culex mosquitoes fed solely on birds, and many mosquito species are thought to have fed on birds within the facility. Particularly, Cx. pipiens group probably fed on both rescued and free-living birds. The rehabilitation facility may be an important site for the introduction and spread of pathogens because 1) numerous mosquitoes inhabit the hospital and its surroundings; 2) blood-fed mosquitoes are caught within the hospital; 3) there is direct contact between birds and mosquitoes; 4) both birds within the hospital and wild birds are fed upon. Furthermore, blood-fed Cx. pipiens form molestus were observed in the winter, suggesting that pathogens might be transmitted even during the winter when other mosquito species are inactive.

Correction: Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes.

[This corrects the article DOI: 10.1371/journal.pntd.0008986.].

A long-term field study on mosquito vectors of avian malaria parasites in Japan.

Avian malaria is a mosquito-borne disease of birds caused by avian Plasmodium spp. in worldwide scale. Some naïve birds show serious symptoms which can result in death. Surveillance of vectors and parasites are important to understand and control this disease. Although avian malaria has been found in Japan, detailed prevalence and dynamics remained understudied. We aimed to observe annual changes in the abundance of mosquitoes and the prevalence of avian Plasmodium parasites in Japan. Mosquitoes were collected using dry ice traps over a 10-year period, at a fixed research area located in Kanagawa prefecture. Collected mosquitoes were investigated for the species composition, population size and prevalence of avian Plasmodium by PCR. Mosquitoes belonging to 13 species in 7 genera were collected (n=8,965). The dominant species were Aedes (Ae.) albopictus and Culex (Cx.) pipiens group (gr.). Seven avian Plasmodium lineages, all of which were previously known, were detected from Cx. pipiens gr., Ae. albopictus, and Tripteroides bambusa. Three genetic lineages were dominant and were probably transmitted by Cx. pipiens gr. whose could be the primary vector of these parasites. Annual variations in the seasonal prevalence of mosquitoes and avian Plasmodium were revealed for the first time during recent 10 years in Japan. Namely, avian Plasmodium occurrence in the vector population peaked often in June to July and September to October when the density of the vector population was presumably high enough for the transmission of avian Plasmodium upon appearance of infected birds.

Two hidden taxa in the Japanese encephalitis vector mosquito, Culex tritaeniorhynchus, and the potential for long-distance migration from overseas to Japan.

The Culex vishnui subgroups, particularly Culex tritaeniorhynchus, are considered the primary vectors of the Japanese encephalitis virus (JEV) in Asia. Recent molecular phylogenetic analyses of JEV isolates from Asian countries have shown that JEVs with diverse genetic variants are present in Asia. Furthermore, some JEV strains have been found to have crossed the East China Sea and been introduced into Japan. In this study, the possibility of overseas migration of the JE vector mosquito, Cx. tritaeniorhynchus was examined from the genetic, physical, and meteorological perspectives. Molecular phylogenetic analysis was performed based on both whole coding sequences and on the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) gene of Cx. vishnui subgroups collected from Asian countries. Culex tritaeniorhymchus was classified into two genetically independent taxa by COI sequences: the Japanese type (Ct-J), which inhabits Japan except for the Amami Islands of southern Japan, and the continental type (Ct-C), which inhabits the Asian region except for Japan. It was confirmed that approximately 10% of Cx. tritaeniorhynchus trapped during the summer in western Kyushu were Ct-C, and that they could fly for up to 38 h continuously. The meteorological analysis also confirmed that the atmospheric flow occurring over the continent coincided with the date of Ct-C capture. This is the first report showing the existence of two taxa in Cx. tritaeniorhynchus. Their physical and physiological characteristics suggest the possibility of long-distance migration from overseas regions to Japan across the East China Sea. Future efforts are expected to provide evidence to support the occurrence of long-distance migration of Cx. tritaeniorhynchus with JEV.

Population genetics of an invasive mosquito vector, Aedes albopictus in the Northeastern USA.

The Asian tiger mosquito (Aedes albopictus) arrived in the USA in the 1980's and rapidly spread throughout eastern USA within a decade. The predicted northern edge of its overwintering distribution on the East Coast of the USA roughly falls across New York, Connecticut, and Massachusetts, where the species has been recorded as early as 2000. It is unclear whether Ae. albopictus populations have become established and survive the cold winters in these areas or are recolonized every year. We genotyped and analyzed populations of Ae. albopictus from the northeast USA using 15 microsatellite markers and compared them with other populations across the country and to representatives of the major global genetic clades to investigate their connectivity and stability. Founder effects or bottlenecks were rare at the northern range of the Ae. albopictus distribution in the northeastern USA, with populations displaying high levels of genetic diversity and connectivity along the East Coast. There is no evidence of population turnover in Connecticut during the course of three consecutive years, with consistent genetic structure throughout this period. Overall, these results support the presence of established populations of Ae. albopictus in New York, Connecticut, and Massachusetts, successfully overwintering and migrating in large numbers. Given the stability and interconnectedness of these populations, Ae. albopictus has the potential to continue to proliferate and expand its range northward under mean warming conditions of climate change. Efforts to control Ae. albopictus in these areas should thus focus on vector suppression rather than eradication strategies, as local populations have become firmly established and are expected to reemerge every summer.

Updated distribution of anopheline mosquitoes in Hokkaido, Japan, and the first evidence of Anopheles belenrae in Japan.

BACKGROUND: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. METHODS: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. RESULTS: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. CONCLUSION: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.

Vector incrimination and transmission of avian malaria at an aquarium in Japan: mismatch in parasite composition between mosquitoes and penguins.

BACKGROUND: Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultaneous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. METHODS: Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mosquitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confirmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identified using molecular methods. RESULTS: Parasite detection in penguins tended to fluctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plasmodium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. CONCLUSIONS: The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confirm competency and infection dynamics of avian malaria parasites.

Discovery of a Novel Flavivirus (Flaviviridae) From the Horse Fly, Tabanus rufidens (Diptera: Tabanidae): The Possible Coevolutionary Relationships Between the Classical Insect-Specific Flaviviruses and Host Dipteran Insects.

Tabanid flies (Tabanidae: Diptera) are common hematophagous insects known to transmit some pathogens mechanically or biologically to animals; they are widely distributed throughout the world. However, no tabanid-borne viruses, except mechanically transmitted viruses, have been reported to date. In this study, we conducted RNA virome analysis of several human-biting tabanid species in Japan, to discover and characterize viruses associated with tabanids. A novel flavivirus was encountered during the study in the Japanese horse fly, Tabanus rufidens (Bigot, 1887). The virus was detected only in T. rufidens, but not in other tabanid species, and as such was designated Tabanus rufidens flavivirus (TrFV). TrFV could not be isolated using a mammalian cell line and showed a closer phylogenetic relationship to the classical insect-specific flaviviruses (cISFs) rather than the vertebrate-infecting flaviviruses (VIFs), suggesting that it is a novel member of the cISFs. The first discovery of a cISF from Brachycera provides new insight into the evolutionary history and dynamics of flaviviruses.

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.

Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes.

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world's most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species' vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector's competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%-16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.

Elevational Distribution of the Asian Tiger Mosquito, Aedes albopictus, in the Inland Mountain Area of Nagano and Yamanashi Prefectures, Japan.

Nagano and Yamanashi prefectures of Japan comprise an inland mountain area that extends widely north and south, with elevation varying greatly by location. Mosquitoes transmitting infectious disease have a diversity of habitats in Nagano and Yamanashi, and many species can be expected there. However, there have been few reports on mosquito fauna; in particular, little information is available on mosquitoes such as Aedes albopictus. The mosquito fauna was investigated to clarify their elevational distribution ranging from 317 to 1,534 m, focusing especially on Ae. albopictus, in 24 areas in Nagano and Yamanashi prefectures from July to September in 2012-16. Adult mosquitoes were collected using the Centers for Disease Control and Prevention traps in each area, in addition to larval mosquito collections at several temple and shrine precincts in each area. At sites where elevations were >800 m (14 sites), no adult Ae. albopictus were captured. In addition, larval Ae. albopictus were not collected at elevations >728 m (15 sites). Aedes albopictus was captured at 20 other sites (annual mean air temperature ranged from 9.4 to 15.2°C, July-September mean air temperature ranged from 19.9 to 25.0°C).

Genetic analysis of Aedes aegypti captured at two international airports serving to the Greater Tokyo Area during 2012-2015.

The introduction of exotic disease vectors into a new habitat can drastically change the local epidemiological situation. During 2012-2015, larvae and an adult of the yellow-fever mosquito, Aedes aegypti, were captured alive at two international airports serving the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from overseas via air-transportation. To infer the places of origin of those mosquitoes, we genotyped the 12 microsatellite loci for which the most comprehensive population genetic reference is currently available. Although clustering by Bayesian and multivariate methods both suggested that all those mosquitoes captured at the airports in Japan belonged to the Asia/Pacific populations, they were not clustered into a single cluster. Moreover, there was variation in mitochondrial cytochrome oxidase I gene (CoxI) haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. We conclude there is little evidence to support the overwintering of Ae. aegypti at the airports; nevertheless, special attention is still needed to prevent the invasion of this prominent arbovirus vector.

Deciphering the Virome of Culex vishnui Subgroup Mosquitoes, the Major Vectors of Japanese Encephalitis, in Japan.

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.

First detection of a Vssc allele V1016G conferring a high level of insecticide resistance in Aedes albopictus collected from Europe (Italy) and Asia (Vietnam), 2016: a new emerging threat to controlling arboviral diseases.

IntroductionAedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission.AimTo determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions.MethodsWe sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel (Vssc) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed.ResultsMost populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance (kdr) allele V1016G in Vssc for the first time in Ae. albopictus. Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S.ConclusionThe V1016G allele was detected in bothAsian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus, particularly where this mosquito species is the main vector of arboviruses.

Morphological and molecular evidence for a new species of Lutzia (Diptera: Culicidae: Culicini) from Thailand.

We found a species of LutziaTheobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological and DNA sequence differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances 3.9-5.6, and 5.1-6.6, respectively). However, the two gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2-2.4%). However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.

Dengue Virus Infection in Aedes albopictus during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan.

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.

Culex (Culiciomyia) sasai (Diptera: Culicidae), senior synonym of Cx. spiculothorax and a new country record for Bhutan.

Culex (Culiciomyia) spiculothorax was described from Thailand based on the presence of spiculation on the thorax of larvae. Adult females are characterized but are indistinguishable from those of related species, such as Cx. pallidothorax. Phylogenetic analysis of mitochondrial oxidase subunit I (COI) sequences revealed that specimens identified as Cx. spiculothorax from Thailand, Japan and Bhutan form a single clade with Cx. sasai from Japan (Kimura 2-parameter genetic distances 0-0.9%) that is clearly distinct from clades comprised of other species of subgenus Culiciomyia. Attempts to collect Cx. sasai from several locations in Japan were unsuccessful - only larvae with thoracic vesicular-like spicules identified as Cx. spiculothorax were collected. Careful examination of specimens collected near the type locality of Cx. sasai revealed the presence of spicules on the thorax. Based on these findings, Cx. spiculothorax is formally synonymized with Cx. sasai, which replaces the former as the species present in Thailand and is a new country record for Bhutan.

Complete Genome Sequencing and Phylogenetic Analysis of a Getah Virus Strain (Genus Alphavirus, Family Togaviridae) Isolated from Culex tritaeniorhynchus Mosquitoes in Nagasaki, Japan in 2012.

Getah virus (GETV; genus Alphavirus, family Togaviridae) is a mosquito-borne virus known to cause disease in horses and pigs. In 2014, for the first time in ∼30 years, a sudden GETV outbreak occurred among racehorses in Ibaraki, Japan. Two years before this outbreak, we obtained multiple GETV isolates from Culex tritaeniorhynchus mosquitoes collected in Nagasaki, Japan and determined the whole genome sequence of GETV isolate 12IH26. Our phylogenetic analysis of GETV strains revealed that the isolate 12IH26 forms a robust clade with the epidemic strains 14-I-605-C1 and 14-I-605-C2 isolated from horses in the 2014 outbreak in Ibaraki. Furthermore, the complete genomic sequence of the isolate 12IH26 was 99.9% identical to those of the 2014 epidemic strains in Ibaraki. Phylogenetic analysis also showed that the recent Japanese GETV strains, including the isolate 12IH26, are closely related to the Chinese and South Korean strains rather than the previous Japanese strains, suggesting that GETV strains may be transported from overseas into Japan through long-distance migration of the infected mosquitoes or migratory birds.

Multiple QTL Determine Dorsal Abdominal Scale Patterns in the Mosquito Aedes aegypti.

The mosquito, Aedes aegypti (L.) originated in Sub-Saharan Africa as a dark form sylvan species (A. aegypti formosus). Evolution of A. aegypti aegypti type form as a human commensal facilitated its colonization of most semitropical and tropical areas. We investigated the genetic basis for abdominal white scale presence that represents the diagnostic for sylvan A. aegypti formosus (scales absent), from type form (scales present) and A. aegypti queenslandensis form (dense scaling). We performed quantitative trait locus (QTL) mapping using 3 criteria for scale patterns among 192 F1 intercross progeny from matings between a queenslandensis type and an aegypti type form. Results identified 3 QTL determining scale patterns and indicated that classification criteria impact robustness of QTL LOD support. Dark- and light-colored forms exist in sympatry, but vary in multiple phenotypic characteristics, including preferences for vertebrate host, oviposition container, house-entering behavior, and dengue vector competence. Markers associated with 2 QTL regions reflected major reductions in recombination frequencies compared with the standard type form linkage map, suggestive of inversion polymorphisms associated with observed linkage disequilibrium between type-specific characteristics. Understanding the genic basis for differences in A. aegypti forms could inform efforts to develop new mosquito and arboviral disease control strategies.