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There are three main innate immune mechanisms against viruses in mosquitoes. Infection with the flavivirus dengue virus is controlled by RNA interference (RNAi) and the JAK-STAT and Toll signaling pathways. This study showed that another flavivirus, Japanese encephalitis virus (JEV), did not invade the salivary glands of Aedes aegypti and that this may be a result of the innate immune resistance to the virus. Argonaute 2 (Ago2) plays a critical role in the RNAi pathway. To understand the mechanism of JEV resistance, we focused on Ago2 as a possible target of JEV. Here, we show that the expression of MyD88 (a mediator of Toll signaling) and Ago2 mRNAs was induced by JEV in the salivary glands of Ae. aegypti mosquitoes and that Ago2, JAK, and domeless (DOME) mRNAs were induced by JEV in the bodies of Ae. aegypti mosquitoes. Double-stranded (ds) Ago2 RNA enhanced JEV infection, and the virus was detected in salivary glands by immunofluorescence assay. In contrast, MyD88 dsRNA had no effect on JEV infection. These data suggest that Ago2 plays a crucial role in mediating the innate immune response of Ae. aegypti to JEV in a manner similar to that employed by dengue virus.

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.

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.

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.

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.

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.