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Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

A German traveler developed dengue fever in late August 2013, following a direct flight from Germany. Autochthonous dengue virus (DENV) infection has not been reported in Japan. To evaluate the risk of autochthonous DENV transmission in Japan, the authors performed a retrospective search of the five areas visited by the German patient to determine the population density of dengue vector mosquito, Aedes albopictus. The annual mean temperature of each area was higher than 12°C, which is considered suitable for the establishment of A. albopictus populations. Our retrospective search revealed the population density of A. albopictus to be high in the urban areas of Japan., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4'-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

The mosquito Aedes albopictus, indigenous to Southeast Asia and nearby islands, has spread almost worldwide during recent decades. We confirm the invasion of this mosquito, first reported in Yamagata city in northeast Honshu, Japan in 2000. Previously, only Ae. japonicus japonicus had been collected in this place, but 2 years later, the population of Ae. albopictus had increased, so more than 80% of the total number of larval colonies there consisted of this species. In contrast to Yamagata's new residential area, now infested by Ae. albopictus, the original mosquito remains in the city but its habitats are generally closer to the surrounding mountains, where the normalized difference vegetation index is higher. The factors affecting the distribution of both species in Yamagata city were studied using geographical information systems (GIS) based on data derived from field surveys, aerial photographs, satellite images and digital maps. The range of Aedes mosquito habitats was estimated and visualised on polygon maps and no significant differences were noted when the polygon area was calculated by GIS software in comparison with the satellite images. Although Ae. j. japonicus was expected to be rapidly overrun by Ae. albopictus, this did not happen. Currently, both species coexist; not only in separate sites, but also simultaneously in various water bodies, where larvae from both species have frequently been seen. However, the competitive relationship between these two Aedes species within a warming environment is an issue that should be closely monitored.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

It is difficult to distinguish infections with different Bartonella species using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis of Bartonella quintana infections, we investigated the antigenicity of B. quintana proteins using sera from homeless people with high B. quintana IgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected with B. quintana reacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis of B. quintana infections from other Bartonella infections.

Japanese encephalitis virus (JEV) infection in mosquitoes was monitored in Vietnam from 2006 to 2008. A total of 15,225 mosquitoes, identified as 26 species in five genera were collected and 12,621 were grouped into 447 pools for examination of JEV infection by assays for cytopathic effects in C6/36 cells and by RT-PCR to detect flavivirus RNA. Three JEV strains were isolated from Culex tritaeniorhynchus Giles collected in northern and southern Vietnam and two JEV strains were isolated from Culex vishnui Theobald collected in the highlands of Vietnam. Genetic and phylogenetic analyses, based on complete E gene nucleotide sequences, revealed that the five JEV strains were classified into the genotype I group and six amino acid differences were found in these five strains. These results indicated that multiple JEV genotype I populations are circulating countrywide in Vietnam, transmitted by bites of their Cx. tritaeniorhynchus and Cx. vishnui.

Calliphora nigribarbis Vollenhoven is a possible mechanical transmitter of highly pathogenic avian influenza. Based on laboratory tests, we evaluated the efficacy of a long-lasting permethrin-treated mosquito netting, known as the Olyset net, for the prevention of this species entering livestock barns. Flies were trapped in Olyset net cages, and two statistics for knockdown and lethal efficacies were obtained. Median knockdown time in the cage (KT50) was estimated to be 341 s for females, and median lethal time after exposure to the mesh (LT50) was estimated to be 30 s and <15 s for females and males, respectively. These LT50s were faster than those measured for anesthetized stationary flies brought in contact with the Olyset net (> 120 s for both sexes),indicating that a fly's spontaneous contact with the Olyset net accelerates insecticide adhesion. The rate of permethrin adhesion to C. nigribarbis after its spontaneous contact with the Olyset net was estimated to be 3.7 ng/s for females, in reference to the 50% lethal dose (LD50) value (112 ng/female), which was obtained from the topical application bioassay of permethrin. The lethality exhibited after brief spontaneous contact with the Olyset net suggests its potential utility in poultry farms against C. nigribarbis invasion.

We established a continuous cell line from the embryo of the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae), a known major vector of the Japanese encephalitis virus (family Flaviviridae, genus Flavivirus) in Asia. The cell line, designated NIID-CTR, was serially subcultured in VP-12 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS). It continued to grow for more than 60 passages over a 750-d period. The NIID-CTR cell line mainly comprised two morphologically distinct types of cells with adhesive properties: spindle-shaped and round cells. Most of the NIID-CTR cells at the 45th passage were diploid (2n = 6). The growth kinetics of the NIID-CTR cells was significantly affected by the FBS concentration in the medium. The population doubling time of the NIID-CTR cells was 20 h in the presence of 10 % FBS and 76 h in its absence. The DNA sequence of the mitochondrial cytochrome oxidase I gene confirmed that the NIID-CTR cell line was derived from C. tritaeniorhynchus. The cells were highly susceptible to Japanese encephalitis and Dengue viruses, thus providing a valuable tool for the study of mosquito-borne flaviviruses.

Culex flavivirus (CxFV) is an insect-specific flavivirus that has recently been detected in various Culex spp. mosquitoes worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of a Tokyo strain, CxFV-NIID21. The full-length CxFV-NIID21 cDNA was cloned into the low-copy-number plasmid pMW119, which was stably amplified in Escherichia coli. Transfection of a mosquito cell line with in vitro-transcribed RNA from the cDNA clone resulted in the production of recombinant progeny virus with growth properties, cytopathogenicity, and virion morphology similar to the parental virus.

Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

The 2003-2004 H5N1 highly pathogenic avian influenza (HPAI) outbreaks in Japan were the first such outbreaks in 79 years in Japan. Epidemic outbreaks have been occurring in Southeast Asia, with the most recent in 2010. Knowledge of the transmission route responsible for the HPAI outbreaks in these countries remains elusive. Our studies strongly suggested that field and laboratory studies focusing on mechanical transmission by blow flies should be considered to control H5N1 avian influenza outbreaks, in particular in epidemic areas, where there are high densities of different fly species throughout the year. In this paper, we review these field and laboratory entomological studies and discuss the possibility of blow flies transmitting H5N1 viruses.

Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The Asian tiger mosquito, Aedes albopictus (Skuse), is the major vector of Chikungunya fever and the secondary vector of dengue fever. We collected Ae. albopictus from Singapore and performed genotyping assay to detect mutations of the voltage-gated sodium channel, which is the target site of pyrethroid insecticides. We detected an amino acid substitution, F1534C, which is suspected to confer knockdown resistance (kdr) to pyrethroid insecticides. Of the collected mosquitoes, 53.8% were homozygous for this mutation, and the allele frequency of this mutation was estimated to be 73.1%. No kdr mutation was detected in the 5 other loci of domains II and IV. This is the first evidence for the presence of the kdr gene in Ae. albopictus, and our findings highlight the need for studying the global distribution of this allele in this important vector insect.

A laboratory colony of the mosquito Aedes japonicus japonicus, which has recently invaded the United States and is recognized as a highly competent vector of West Nile virus, was established from larvae collected in Narita, Japan. The mosquitoes were maintained with induced insemination, blood-feeding on humans, and oviposition in water provided from the original collection site during the first few generations, then the colony was transferred to a large cage (40×40×100 cm in height) and adapted to conditions in which specimens were allowed to mate freely. White mice were provided as the blood source, and deionized water was available for oviposition. Approximately 185 eggs, most of which were tolerant to desiccation for at least 1 month, with some surviving for up to 2.5 months, were obtained per female following a single blood-feeding. The rate of successful emergence was nearly 90%, although this rate decreased significantly at high larval densities. The colony has been maintained for 5 years, and developmental profiles of the species have been obtained during that time.

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.

To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003-2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pallens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.

We conducted laboratory experiments to verify molecular techniques of avian malaria parasite detection distinguishing between an infected mosquito (oocysts on midgut wall) and infective mosquito (sporozoites in salivary glands) in parallel with blood-meal identification from individual blood-fed mosquitoes prior to application to field survey for avian malaria. Domestic fowl infected with Plasmodium gallinaceum was exposed to a vector and non-vector mosquito species, Aedes aegypti and Culex pipiens pallens, respectively, to compare the time course of polymerase chain reaction (PCR) detection for parasite between competent and refractory mosquitoes. DNA of the domestic fowl was detectable for at least 3 days after blood feeding. The PCR-based detection of P. gallinaceum from the abdomen and thorax of A. aegypti corresponded to the microscopic observation of oocysts and sporozoites. Therefore, this PCR-based method was considered useful as one of the criteria to assess developmental stages of Plasmodium spp. in mosquito species collected in the field. We applied the same PCR-based method to 21 blood-fed C. sasai mosquitoes collected in Rinshi-no-mori Park in urban Tokyo, Japan. Of 15 blood meals of C. sasai successfully identified, 86.7% were avian-derived, 13.3% were bovine-derived. Plasmodium DNA was amplified from the abdomen of three C. sasai specimens having an avian blood meal from the Great Tit (Parus major), Pale Thrush (Turdus pallidus), and Jungle Crow (Corvus macrorhynchos). This is the first field study on host-feeding habits of C. sasai in relation to the potential role as a vector for avian malaria parasites transmitted in the Japanese wild bird community.

We isolated a new flavivirus from Aedes albopictus mosquito and a related species in Japan. The virus, designated Aedes flavivirus (AEFV), only replicated in a mosquito cell line and produced a mild cytopathic effect. The AEFV genome was positive-sense, single-stranded RNA, 11,064 nucleotides in length and contained a single open reading frame encoding a polyprotein of 3341 amino acids with 5' and 3' untranslated regions (UTRs) of 96 and 945 nucleotides, respectively. Genetic and phylogenetic analyses classified AEFV with the insect flavivirus, but distinct from Cell fusing agent (CFA), Kamiti river virus and Culex flavivirus. Interestingly, a partial sequence of AEFV showed significant similarity to that of Cell silent agent (CSA), the insect flavivirus-related nucleotide sequence integrated in the genome of A. albopictus. These results suggest that AEFV is a new member of the insect flaviviruses, which are intimately associated with Aedes mosquitoes and may share a common origin with CSA.

In a previous study, the highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from blow flies collected at the Tamba Town of Kyoto prefecture during the outbreak period in March 2004. In this study, we carried out virus exposure experiments to investigate whether the H5N1 virus would survive in a blow fly, Calliphora nigribarbis. The virus exposure experiments showed that the H5N1 influenza virus was isolated from the crop and intestine of C. nigribarbis for at least 24 h, and the viruses remained viable with titers ranging from 0.5 to 4.63 TCID50. This result suggests that C. nigribarbis could possibly transport the H5N1 virus over a distance of 2 km, which is the distance they can migrate within 24 h.

A mark-release-recapture study of the dispersal ability of blow flies, Calliphora nigribarbis, was conducted in Ikumo-Naka, Ato Town, Yamaguchi Prefecture, Japan in December 2004. A location where a fatal avian influenza outbreak had occurred 1 year previously was selected for the present study. A total of 3,884 C. nigribarbis were collected, 1,915 of which were marked and released from 4 different collection sites during 2 successive days. The recapture rate of the released C. nigribarbis ranged from 0.014 to 0.029 among the collection sites, and the overall recapture rate was calculated as 0.022. Based on the distance between the released site and the recaptured site, the dispersal rate of C. nigribarbis was estimated as 256 m/h on the 1st day and 179 m/h on the 2nd day of the experiment, and the maximum dispersal rate observed in this study was estimated as 500 m/h. Taking into account the active period of C. nigribarbis on a fine day (7 h/day), the distance traveled by C. nigribarbis within a day was estimated as 1,789 and 1,250 m/day on average for the 1st and 2nd days, respectively, and the maximum distance was 3,500 m/day. These results suggest that C. nigribarbis could play a role in the mechanical dissemination of avian influenza virus and spread of the outbreak in Japan.

In order to develop an inexpensive, simple, and accurate method of monitoring for the reemergence of schistosomiasis japonica in Yamanashi Prefecture, Japan, the distribution and habitation density of the intermediate host, Oncomelania nosophora, were spatially analyzed using geographic information systems. The 1967-1968 density distribution maps prepared by Yamanashi Prefecture and Nihei were digitized and geocoded. The habitats and population density of O. nosophora were estimated by referring to the data compiled by the Yamanashi Association for Schistosomiasis Control (1977). These earlier findings were compared with average population densities between 1996 and 2000 previously recorded (Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 52, 395-401, 2003 and Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 53, 199-205, 2004). A variance map was created to compare the spatial distribution maps of population density from each of the two periods of interest. The changes in distribution were remarkable and the map was found to be effective for future control. The most appropriate monitoring sites were chosen on the basis of the spatial population density maps and the variance map. Moreover, the paddy fields at risk were extracted using the normalized difference vegetation index value based on Advanced Land Observation Satellite images. The combination of this method with the global positioning system provides an inexpensive means of monitoring modern schistosomiasis endemic areas in Japan and also in China, the Philippines, and other countries as well, where the intermediate snail grows in paddy fields and marshlands under consistently wet conditions.

A mark-release-recapture of Culex pipiens pallens was conducted in an urban area of Japan during June 26-29, 2007. Larvae naturally occurring in rain gutters were collected and reared to adults in a laboratory. A total of 10,183 emerging female Cx. pipiens pallens of 4-8 days old were marked with fluorescent dye and released from one site. Recapture was made on 4 consecutive days using 41 Centers for Disease Control and Prevention traps with 1 kg of dry ice and human landing collection, and 121 marked females were recaptured. The overall recapture rate was 0.01. The mean distance traveled by the recaptured females was estimated as 470, 287, 326, and 517 m on days 1-4, respectively. The maximum flight distance of host-seeking Cx. pipiens pallens was estimated as 1,217 m based on the relationship between distance from the release site to the collection site and the total number of recaptures/traps. The population size of female Cx. pipiens pallens in the study area was estimated as 100,904 +/- 8,509. The size of operational area for the control of Cx. pipiens pallens in urban area is discussed.

The Culex pipiens complex consists of vector mosquitoes that transmit important human pathogens. In this study we established a simplified method to distinguish three members of the Cx. pipiens complex, Cx. p. pallens Coquillet, Cx. p. form molestus Forskal, and Cx. quinquefasciatus Say, collected in Japan. Sequence analysis of the Drosophila Ace-orthologous acetylcholinesterase (Ace) gene (668 to 680 bp) revealed that a single polymorphic region characterizes each species. Based on this region, specific primers that distinguish Cx. p. form molestus (ACEpip2) and Cx. p. pallens (ACEpall2) were newly designed. Polymerase chain reactions were performed with the genomic DNA of Culex mosquitoes as the template, and these primers clearly distinguished two Culex spp. The accuracy of the designed primers was evaluated with 38 colonies of mosquito samples collected from 9 prefectures of Japan. The testing revealed that the distribution of anautogenous Cx. p. pipiens has not been confirmed in Japan. It also revealed that the male of Cx. p. pallens possesses an Ace gene haplotype that is highly similar to the sequence of Cx. quinquefasciatus. This improved method allows the evaluation of vector competence of Cx. p.molestus, which is the suspected vector of West Nile virus.

Culex pipiens complex is the significant vector mosquito of West Nile virus. To take stock of the current situation of insecticide susceptibilities and design an ideal mosquito control strategy, we collected Culex pipiens pallens Coquillet, Culex pipiens form molestus Forskal, and Culex quinquefasciatus Say from fields in Japan and conducted bioassays for five larvicides (fenitrothion, temephos, etofenprox, diflubenzuron, and pyriproxyfen) by using a larval dipping method. Among five insecticides tested, obvious reduced susceptibilities were observed for etofenprox, which is the only pyrethroid compound registered as a larvicide in Japan. Twenty-two of 56 colonies exhibited a >10% survival rate at the etofenprox concentration of 5.7 microg/ml, which is a 10 times higher concentration of the working solution. The LC50 of a colony collected from Fukuoka prefecture for etofenprox exceeded 60 microg/ml (resistance ratio >2,307), and this colony also exhibited cross-resistance to other pyrethroids, permethrin (299-fold) and phenothrin (1,200-fold). The insect growth regulators diflubenzuron and pyriproxyfen were found to be sufficiently effective enough to control Culex larvae present, but decreased sensitivities to these insecticides were slightly detected in some colonies of Cx. p. form molestus collected from urban areas. Several etofenprox-resistant colonies of Cx. p. form molestus exhibited simultaneously decreased susceptibilities to other insecticides, including temephos, diflubenzuron, and pyriproxyfen.

Although body louse is a well-known vector of trench fever, the growth kinetics of Bartonella quintana in body lice has not been fully understood. We performed a quantitative analysis of bacterial multiplication rate. B. quintana started proliferation in body lice 4 days after ingestion and was constantly excreted in the feces for at least 3 weeks. The number of bacteria in feces reached the maximum 10(7)/louse per day on Day 15. The doubling time of B. quintana estimated from logistic regression formula was 21.3 hours. Scanning electron microscopy showed the presence of bacterial masses in feces. Immunofluorescent study using specific monoclonal antibody confirmed identification of B. quintana. Such an explosive multiplication rate and active excretion of B. quintana from the body lice could be related to epidemics of trench fever in developed countries.

This study on the ultrastructure of the oocysts of four isolated species of Ascogregarina (A. taiwanensis (Lien and Levine) (Eugregarinidae: Lecudinidae) from Aedes albopictus (Skuse), A. culicis (Ross) (Eugregarinidae: Lecudinidae) from Aedes aegypti (L.), A. armigerei (Eugregarinidae: Lecudinidae) from Armigeres subalbatus (Coquillet), and Ascogregarina sp. (Eugregarinidae: Lecudinidae) from Ochlerotatus japonicus japonicus (Theobald)) using a scanning electron microscope revealed significant differences in size and in surface structure. The average length of the oocyst was greatest in A. armigerei (13.2+/-0.2 microm) (mean+/-SD) and least in A. culicis (8.8+/-0.4 microm). Oocysts were of moderate length in A. taiwanensis (9.9+/-0.6 microm) and in Ascogregarina sp. (10.7+/-1.1 microm) isolated from O. j. japonicus. The ultrastructure of the surface of the A. culicis oocyst was rough in texture with numerous dense spots and was easily distinguishable from the oocysts of the other three Ascogregarina spp. The maximum likelihood tree inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicated that the four Ascogregarina spp. form a monophyletic cluster among other gregarine parasites. Within the Ascogregarina clade, A. culicis, A. taiwanensis, and Ascogregarina sp. from O. j. japonicus showed a close relationship, whereas A. armigerei was a distantly related species.

We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.

Two cDNA sequences encoding Drosophila Ace-orthologous and -paralogous acetylcholinesterase precursors (AO- and AP-AChE precursors, respectively), were identified from the body louse, Pediculus humanus humanus L. In vitro inhibition studies with an insecticide-susceptible body louse strain exhibited a simplex inhibitory response of AChE. The I50 values of fenitroxon and carbaryl were estimated to be 2.2 and 1.9 microM for the susceptible lice, respectively. The mRNA level of AP-AChE gene was 3.1- and 9.3-fold higher than that of AO-AChE gene in the abdomen and the combined parts of the head and thorax, respectively, suggesting, due to its abundance, the potential significance of the AP-AChE isoform in Pediculus human lice in association with the efficacy of AChE-targeting pediculicides.

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation "Candidatus Riesia pediculicola" for the louse symbionts.

During the outbreak of highly pathogenic avian influenza that occurred in Tamba Town, Kyoto Prefecture in 2004, a total of 926 flies were collected from six sites within a radius of 2.3 km from the poultry farm. The H5 influenza A virus genes were detected from the intestinal organs, crop, and gut of the two blow fly species, Calliphora nigribarbis and Aldrichina grahami, by reverse transcription-polymerase chain reaction for the matrix protein (M) and hemagglutinin (HA) genes. The HA gene encoding multiple basic amino acids at the HA cleavage site indicated that this virus is a highly pathogenic strain. Based on the full-length sequences of the M, HA, and neuraminidase (NA) segments of virus isolates through embryonated chicken eggs, the virus from C. nigribarbis (A/blow fly/Kyoto/93/2004) was characterized as H5N1 subtype influenza A virus and shown to have > 99.9% identities in all three RNA segments to a strain from chickens (A/chicken/Kyoto/3/2004) and crows (A/crows/Kyoto/53/2004) derived during this outbreak period in Kyoto in 2004. Our results suggest it is possible that blow flies could become a mechanical transmitter of H5N1 influenza virus.

Infection in different stages of larvae of Aedes aegypti and Ae. albopictus with Ascogregarina taiwanensis and A. culicis, respectively, revealed that the oocysts of Ascogregarina spp. are able to infect any instar and can complete their life cycle within 9.5 +/- 1 days. When early instars ingested oocysts, parasite development was synchronized to larval-pupal ecdysis and oocyst dissemination occurred at the time of adult emergence, oviposition, or both. The parasites also developed normally when infecting 2nd, 3rd, and early 4th instars and oocysts were released only during oviposition. The parasitic development stopped at the gamont stage when oocysts were ingested by late 4th instars (6 days old). The release of sporozoites in the midgut of any larval stage started within 45 min of oocyst ingestion. About 98% of oocysts of both A. taiwanensis and A. culicis were emptied within 2-3 h of their ingestion in their respective hosts. The oocysts of both species remained viable on desiccated filter paper stored at 27 degrees C and 65 +/- 5% relative humidity, indicating that the oocysts were resistant to dryness. The oocysts of A. culicis could survive up to 6 months, whereas those of A. taiwanensis survived up to 4 months. These biological characteristics relating to parasite development might enhance the distribution of Ascogregarina spp. widely in nature and facilitate the species to be considered for biological control of Aedes mosquitoes in the future.

In an epidemiological investigation of trench fever in Japan, we compared the seroprevalence of Bartonella quintana in homeless people and in the general population. In homeless rescue outreach programs held in Tokyo from May 2001 to March 2003, 151 blood samples were taken from non-hospitalized homeless people. The prevalence of IgM and IgG antibodies against B. quintana in these people was compared with that in 200 healthy blood donors using a commercially available indirect fluorescent antibody test. Although IgG titers of > or = 1:128 were found in 57% (86/151) of homeless people and 51% (101/200) of blood donors, high titers of > or = to 1:1,024 were encountered only in homeless people (11%, 16/151). Attempts to isolate B. quintana from the blood of homeless people were unsuccessful, but polymerase chain reaction based detection, using Bartonella genus specific primers, demonstrated the presence of B. quintana DNA in the blood of 10 homeless people. Our data suggest that urban trench fever is endemic among the Japanese homeless population.

Trench fever is a body louse-borne disease caused by Bartonella quintana Brenner. The recent status of louse infestation in Nepalese children is not well known. We collected head and body lice, Pediculus humanus capitis De Geer and Pediculus humanus humanus L., respectively, from 30 children, including 11 cases of double infestation with both head and body lice. Detection of B. quintana in both louse species identified was carried out by polymerase chain reaction (PCR). PCR products with B. quintana DNA sequences were detected in both head and body lice from two children as well as in body lice derived from two other children. These results demonstrate that head lice may also play a role in the transmission of trench fever.

A new, convenient method was developed to supply CO2 for mosquito sampling by using yeast, which converts sugar into CO2 and ethyl alcohol. The system could, at average, generate 32.4 ml/min of CO2 for at least 27 h. The total weight of the CO2 generated was estimated to be 94 g. The efficacy of yeast-generated CO2 as attractant for mosquitoes was significant, and the following 6 mosquito species were collected using yeast-generated CO2 traps from July to September 2003 in a residential area of southern and northern Yokohama City, Japan: Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), Culex halifaxii Theobald, Cx. pipiens pallens Coquillett, Ochlerotatus japonicus (Theobald), and Tripteroides bambusa (Yamada). Besides mosquitoes, various other insects were collected in the trap. Species compositions of insects collected in yeast-generated CO2 traps and dry-ice-baited traps were compared.

We applied remote sensing using satellite images capable of obtaining data over a broad range, transcending national borders, as a method of rapidly, precisely, and safely increasing our understanding of the potential distribution of malaria. Our target region was the so-called Mekong malaria region on the Indochina Peninsula. As a malaria index, we used existing distribution maps of total reported malaria cases, malaria mortality, vivax malaria and falciparum malaria incidences, and so forth for 1997 and 1998. We produced monthly distribution maps of a normalized difference vegetation index (NDVI) with values of 0.2+, 0.3+, 0.35+, and 0.4+ using the geographical information system/remote sensing software based on the East Asia monthly NDVI maps of 1997. These maps were overlaid with various malaria index distribution maps, and cross-tabulations were carried out. The resulting maps with NDVI values of 0.3+ and 0.4+ matched the falciparum malaria distribution well, and we realized, in particular, that falciparum malaria is prevalent in regions in which NDVI values of 0.4+ continue for 6 months or more, while cases are fewer in regions with NDVI values of 0.4+ that continue for 5 months or less. It will be necessary in the future to examine the relationship between NDVI values and the habitats of the various vector mosquitoes using high-resolution satellite images and to implement detailed forecasts for malaria endemic areas by means of NDVI.