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Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes -borne infectious diseases.

BACKGROUND Aedes aegypti is a remarkably effective mosquito vector of epidemiologically important arboviral diseases including dengue fever, yellow fever and Zika. The present spread of resistance against pyrethroids, the primary insecticides used for mosquito control, in global populations of this species is of great concern. The voltage‐gated sodium channel (VGSC) in the nervous system is the known target site of pyrethroids in insects. Past studies have revealed several amino‐acid substitutions in this channel that confer pyrethroid resistance, which are known as knockdown resistance (kdr) mutations. RESULTS This study investigated a laboratory colony of Ae. aegypti, MCNaeg, established from larvae collected in Rio de Janeiro, Brazil in 2016. The MCNaeg colony showed strong resistance against pyrethroids without laboratory selection. Of the two VGSC gene haplotypes present within this colony, one harbored three known kdr mutations, V410L, V1016I, and F1534C, and the other harbored only the known F1534C mutation. In latter haplotype, we also found novel amino‐acid substations including V253F. Previous molecular modeling and electrophysiological studies suggest that this residue serves a pyrethroid‐sensing site in the second receptor, PyR2. Our genetical analysis showed that the haplotype harboring V253F and F1534C is associated with equal or slightly stronger resistance than the other triple kdr haplotype to both Type I and Type II pyrethroids. CONCLUSION The novel substitution V253F is potentially involved in pyrethroid resistance in Ae. aegypti. Further studies are needed to elucidate the role of this substitution in the pyrethroid susceptibility of VGSC. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., We found a novel amino‐acid substitution V253F in the voltage‐gated sodium channel in Aedes aegypti which is potentially involved in pyrethroid resistance of this vector mosquito

Aedes aegypti(Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. Nevertheless, the development of pyrethroid resistance is a major obstacle to mosquito/disease control worldwide. Here, we focused on the mutations in the target site of pyrethroid insecticides, voltage-sensitive sodium channel (Vssc), and found thatAe. aegypticollected from Vietnam has the L982W allele in theVsscat a high frequency (>79%). L982W mutation is located in the highly conserved region ofVsscthat is associated with sodium–ion selectivity and permeation rate. Strains having the L982W allele showed similar or even higher levels of resistance to pyrethroids than those having V1016G, a typical knockdown resistance allele in Asia. Furthermore, concomitant mutations L982W+F1534C and V1016G+F1534C were confirmed, and strains having these multipleVsscmutations exhibited incomparably higher levels of pyrethroid resistance than any other field population ever reported. Molecular modeling analysis confirmed that these concomitant mutant alleles could interfere with approaching pyrethroid toVssc. Remarkably, >90% ofVsscofAe. aegyptiwere occupied by these hyper insecticide-resistant haplotypes in Phnom Penh city, Cambodia. Analysis of wholeVssccoding genes suggested thatVsscs have evolved into stronger resistant forms efficiently through gene recombination events. At this point, L982W has never been detected inVsscofAe. aegyptifrom any other neighboring countries. We strongly emphasize the need to be vigilant about these strong resistance genes spreading to the world through Indochina Peninsula.Significance StatementThe high frequency (>78%) of the L982W allele was detected at the target site of the pyrethroid insecticide, the voltage-sensitive sodium channel (Vssc) ofAedes aegypticollected from Vietnam and Cambodia. Haplotypes having concomitant mutations L982W+F1534C and V1016G+F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these haplotypes exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W mutation has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as otherAedes-borne infectious diseases.

Bed bug control highly depends on insecticides with a limited number of modes of action, especially since the global prevalence of pyrethroid resistance. De facto insecticide options against bed bugs in Japan are acetylcholinesterase inhibitors (AChEis) that consist of organophosphates and carbamates. However, the status of AChEi resistance and the mechanisms involved have not been ascertained. An amino acid substitution mutation, F348Y (or F331Y in standard numbering), occurring at an acyl-binding site of the paralogous AChE gene (p-Ace), was identified among AChEi-resistant colonies of both common and tropical bed bugs (Cimex lectularius and C. hemipterus, respectively). This mutation was genetically associated with propoxur and fenitrothion resistance in F348Y-segregating colonies of C. hemipterus. Inhibition of heterologously expressed C. lectularius p-Ace with insecticides revealed that the sensitivities of F348Y-carrying AChE decreased by orders of 10- to more than 100-fold for diazoxon, carbaryl, fenitroxon, paraoxon, chlorpyrifos-methyl, malaoxon, azamethiphos, methyl-paraoxon, and propoxur. In contrast, the mutant AChE showed a slightly decreased degree of sensitivity for dichlorvos and almost unchanged sensitivity for metoxadiazone. Further studies are needed to ascertain whether the practical efficacies of dichlorvos and metoxadiazone are ensured against F348Y-carrying bed bugs and whether other resistance mechanisms are involved.

Aedes albopictus (Skuse) (Diptera: Culicidae) is distributed widely and is common in much of Japan. In Japan, female adults begin to bite in between April and June, except in the southern subtropics where the mosquito has no dormant period. It is difficult to estimate the first Ae. albopictus biting day because it varies annually depending on the location. Over several years, we surveyed the mosquitoes at different locations that covered a range of warmer to cooler areas of Japan. We found an association between the timing of first biting day by Ae. albopictus and spring temperature. In spring months, the strongest correlation was found with mean April temperatures, followed by March. Based on these data, it may, therefore, be possible to apply a simple formula to predict the timing of the first biting day at various geographical locations in Japan. Forecasting maps were created using a simple prediction formula. We found that the first biting day for Ae. albopictus changed depending on early spring temperatures for each year. There was an approximate 20-d difference in first biting day between years with warmer and cooler springs. This prediction model will provide useful insight for planning and practice of Ae. albopictus control programs, targeting larvae and adults, in temperate regions globally., Article, Journal of Medical Entomology.54(6):1519-1524(2017)

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline—MoNaS (Mosquito Na+ channel mutation Search)—which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests., Author summary The Voltage Gated Sodium Channel (VGSC) in insect is targeted by pyrethroid insecticides and genetic variations in the protein are known to confer pyrethroid resistance. Since the VGSC gene in genome consists of many exons and long introns, there is no simple method to genotype whole of coding regions from the genomic DNA of insect. Here, we designed hybridization capture probe set to concentrate VGSC coding exons in NGS library from individual genomic DNA of the arbovirus vector mosquito Aedes albopictus. The probe set we designed was able to capture VGSC exons not only from A. albopictus genomic DNA but also from genomic DNA of two other mosquito species belonging to the same subfamily only with slight decrease of efficiency. The technology will allow unbiased analysis of the VGSC gene in multiple mosquito species with relatively low sequencing cost and enhance discovery of new resistance mutations.

Introduction of exotic diseases 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 in two international airports serving to the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from overseaviaair-transportation. To infer the places of origin of those mosquitoes, we genotyped 12 microsatellite loci for which the most comprehensive population genetic reference is available. Although clustering by Bayesian and multivariate methods both suggested all those airport mosquitoes belong to Asia/Pacific population, they were not clustered into a single population. Also, there was variation in mitochondrialCox1haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. Whereas we conclude there is little evidence to support overwintering ofAe. aegyptiin the airports in this study, special attention is still desired to prevent the invasion of this prominent arbovirus vector.

Tick-borne viruses have emerged recently in many parts of the world, and the discoveries of novel tick-borne viruses have been accelerated by the development of high-throughput sequencing technology. In this study, a cost-efficient small benchtop next-generation sequencer, the Illumina MiniSeq, was used for the RNA virome analysis of questing ticks collected from Hokuriku District, Japan, and assessed for their potential utility in a tick-borne virus surveillance system. We detected two phleboviruses [Kabuto Mountain virus (KAMV) and Okutama tick virus (OKTV)], a coltivirus [Tarumizu tick virus (TarTV)], and a novel iflavirus [Hamaphysalis flava iflavirus (HfIFV)] from tick homogenates and/or cell culture supernatants after virus isolation processes. The number of sequence reads from KAMV and TarTV markedly increased when cell culture supernatants were used, indicating a successful isolation of these viruses. In contrast, OKTV and HfIFV were detected only in tick homogenates but not from cell culture supernatants, suggesting a failure to isolate these viruses. Furthermore, we performed genomic and phylogenetic analyzes of these detected viruses. OKTV and some phleboviruses discovered recently by NGS-based methods were probably deficient in the M genome segment, which are herein proposed as M segment-deficient phlebovirus (MdPV). A phylogenetic analysis of phleboviruses, including MdPV, suggested that Uukuniemi and Kaisodi group viruses and kabutoviruses evolved from an ancestral MdPV, which provides insights into the evolutionary dynamics of phleboviruses as emerging pathogens.

Introduction Aedes 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. Aim To determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions. Methods We 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. Results Most 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. Conclusion The 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.

Introduction Aedes 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. Aim To determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions. Methods We 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. Results Most 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. Conclusion The V1016G allele was detected in both Asian 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.

The objective of this study was to clarify the efficacy of a currently available N,N-diethyl-m-toluamide (DEET) repellent against tick species in Japan. We performed 2 different field trials: "human trap," and "flag-dragging." In total, 482 ticks were collected from white flannel cloths in the field studies. The collected tick species were Ixodes persulcatus and I. ovatus, which accounted for 5.3% and 94.7% of the ticks in the human trap test and 31.4% and 68.6% in the flag-dragging test, respectively. The repellency levels of DEET-treated flannel cloths in the human trap and flag-dragging tests were 84.0% and 99.7%, respectively. The escape times for I. persulcatus and I. ovatus female adults from DEET-treated flannel cloths were determined. The median escape times for I. persulcatus and I. ovatus on DEET-treated flannel cloths were 48 s (95% confidence interval [CI]: 30-96) and 10 s (95% CI: 5-24), respectively. In contrast, many ticks remained on the untreated flannel cloths for 10 min after mounting. These results indicate that DEET repellents appear to prevent tick bites and that the use of DEET repellents against ticks is an effective personal protection measure.

Although the importance of cis-acting mutations on detoxification enzyme genes for insecticide resistance is widely accepted, only a few of them have been determined as concrete mutations present in genomic DNA till date. The overexpression of a cytochrome P450 gene, CYP9M10, is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. The haplotypes of CYP9M10 exhibiting overexpression (resistant haplotypes) belong to one specific phylogenetic lineage that shares high nucleotide sequence homology and the same insertion of a transposable element. Among the resistant haplotypes, allelic progression involving an additional cis-acting mutation and gene duplication evolved a CYP9M10 haplotype associated with extremely high transcription and strong pyrethroid resistance. Here we show that a single nucleotide substitution G−27A, which is located near the transcription start site of CYP9M10, is involved in the progression of the duplicated haplotype lineage. The deletion of a 7-bp AT-rich sequence that includes nucleotide −27 inhibited the initiation of transcription from the original transcriptional initiation site. The mutation was suspected to reside within a core promoter, TATA-box, of CYP9M10.

A method of quantitative elemental analysis of mosquitoes which convey serious infectious diseases, such as Japanese encephalitis and Dengue fever, has been developed in order to clarify their mode of life and sphere of action. The method was developed on the basis of a standard-free method for biological samples. The method enables us to perform quantitative analysis of untreated mosquito. Moreover, the method was successfully applied to quantitative analysis of each part of mosquito's body such as head and chest, abdomen, leg and wing. It was found that there are clear differences among parts in the body of mosquito, and also among different species such as Culex tritaeniorhynchus and Aedes albopictus. The method was successfully applied to quantitative analysis of small fly belonging to Sciaridae (Bradysia.praecox) whose size is smaller than that of mosquito. It was also confirmed that the method is applicable to other kind of bio-samples of small quantities such as a grain of egg of Alaska pollock whose dry weight is less than 100 μg.

Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

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 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.

Summary 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.

Development of insecticide resistance reduces the efficacy of controlling measures against the medical and agricultural insect pests. Cytochrome P450s are one of the major detoxification enzymes involved in insecticide metabolisms. Previously, we have reported that the P450 gene Cyp9m10 is about 260-fold overexpressed in a pyrethroid-resistant strain of Culex quinquefasciatus compared to a susceptible strain. In this study, we obtained direct evidence that the Cyp9m10 overexpression is caused by a cis-acting mutation. Additionally, a region of approximately 100 kb in length including the Cyp9m10 locus was specifically duplicated in the resistant strain. The two duplicated Cyp9m10 copies shared a completely identical sequence within the transcribed region and the flanking region up to the breakpoint located 1.1 kb upstream of the transcriptional start site. A Miniature Inverted-repeat Transposable Element (MITE)-like element was specifically inserted 0.2 kb upstream of both Cyp9m10 copies in the resistant strain. In backcross experiment, a haplotype containing the two duplicated Cyp9m10 copies was strongly associated with the pyrethroid resistance.

Previous studies on a strain of Culex pipiens quinquefasciatus from Saudi Arabia indicated permethrin resistance was a result of cytochrome P450 mediated detoxification and kdr. The P450 detoxification was found to be larval specific and associated with a fitness cost in certain environments. The P450 responsible for resistance (and the fitness cost) has not been identified, but recently two candidate P450s (CYP4H34 and CYP9M10) have been found. We measured cytochrome P450 and cytochrome b₅ content as well as the expression levels of CYP4H34 and CYP9M10 in a susceptible (SLAB) and two isogenic strains (isolated by repeated backcrossing and selection) of mosquito (ISOP450 and ISOJPAL) resistant to permethrin. Cytochrome P450 protein levels of the resistant strains were significantly higher (1.5-fold) than SLAB, but were not significantly different from one another. Expression of CYP4H34 in the larvae and adults of the resistant (ISOP450 and ISOJPAL) and susceptible (SLAB) strains were not statistically different. CYP9M10 was found to be significantly over-expressed in larvae of both permethrin-resistant isogenic strains (1800-fold in ISOP450 and 870-fold in ISOJPAL) when compared to SLAB. Partial sequence analysis of CYP9M10 revealed eight polymorphic sites that distinguished the susceptible allele from the resistant allele. We conclude that CYP9M10 is linked to permethrin resistance in these strains of C. p. quinquefasciatus, and is likely to be the P450 gene responsible for resistance in these strains.

JPal-per strain of Culex quinquefasciatus exhibits extremely high resistance against pyrethroids in larvae, though the resistance is greatly lower in adults. Increased microsome monooxygenase metabolism is one of the major factors of the larval resistance in this strain. We cloned 46 novel cytochrome P450 cDNAs from JPal-per strain. An oligonucleotide microarray was designed for the novel 46 genes plus 16 previously reported P450 genes along with other non-P450 gene probes. Of these, five P450 genes were upregulated (>2.5-fold) in JPal-per larvae as compared with a susceptible strain. The expression ratios for the highest three among the five P450 genes screened in the microarray analysis, CYP9M10, CYP4H34 and CYP6Z10, were further validated by qPCR as 264-, 8.3-, and 3.9-fold, respectively. In JPal-per, the transcription levels of CYP9M10 and CYP4H34 showed a similar stage-dependent pattern as a high expression level during the larvfrom Ogasawara Islands in Japanal stage dramatically decreases in the adult stage. This larval specific overexpression manner of the two genes was consistent with the characteristic of stage-dependent resistance of JPal-per strain previously reported, suggesting that the two P450s, CYP9M10 and CYP4H34, are involved in pyrethroid detoxification in JPal-per strain.

We identified and characterized an alternatively spliced gene that encodes delta glutathione transferases (GSTs) (CqGSTdelta1) in Culex quinquefasciatus. CqGSTdelta1 contains six exons that produce four mature GST transcripts, each sharing exons 1 and 2, and containing a different exon 3 variant (exons 3A, 3B, 3C, or 3D). The exon/intron composition of CqGSTdelta1 showed high similarity with the delta GST genes adgst1AS1 and aggst1α identified from Anopheles dirus and Anopheles gambiae, respectively. Thus, alternative splicing in delta GSTs possibly occurred before the divergence of the Anopheles and Culex mosquitoes. Gene expression analysis clarified that CqGSTd1-1 and CqGSTd1-4 are constitutively and highly expressed in the larvae, pupae, and adults, while CqGSTd1-2 and CqGSTd1-3 are suppressed at all developmental stages, except the pupal stage. Thus, at least two factors may be associated with the transcriptional regulation of these genes. Microarray analysis revealed that none of the genes were overexpressed in a DDT-resistant Cx. quinquefasciatus strain.

Pyrethroid insecticides play very important roles in the control of pediculosis, which is caused by human head louse (Pediculus humanus capitis De Geer) worldwide. The development of head louse resistance to pyrethroid pediculicides has become a global issue during the last two decades. Pyrethroid resistance is associated with amino acid substitutions in the alpha-subunit of the para-sodium channel gene; these substitutions are termed kdr-like mutations. The frequencies of four mutations (D11E, M815I, T929I, and L932F) in the sodium channel gene were studied in human head louse colonies collected from Japan by using SNaPshot method. A total of 630 head lice collected from 282 infested people were analyzed, and it was found that 55 lice of 19 colonies homozygously or heterozygously possessed kdr-like genes, in which the four mutations existed concomitantly. This suggested that the quadruple mutant haplotype is a common feature of the kdr genes of pandemic head lice. The frequencies of the occurrence of resistant genes in the total individuals tested and resistant gene-carrying colonies were 8.7 (55/630) and 6.7% (19/263), respectively. Because the resistant gene was detected in the colonies collected from 11 of the 22 prefectures surveyed, it is speculated that resistant head lice are already spread extensively in Japan. This was the first large-scale survey of pyrethroid resistant head lice in Japan.

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.

Vector-borne diseases result from infections transmitted to humans by blood-feeding arthropods such as mosquitoes, ticks, and fleas. Such cold-blooded animals are influenced by environmental change. A recent IPCC report clearly showed that the emission of greenhouse gases has already changed world climates. Heat waves in Europe, rises in global mean sea level, summer droughts and wild fires, more intense precipitation, and increasing numbers of large cyclones and hurricanes may be typical example of extreme climate phenomena related to global warming. High temperatures may increase survival among arthropods, depending on their vector, behavior, ecology, and valuable factors, and temperate zone warming may accelerate the spread of mosquitoes such asAedes albopictus. The MIROK (K1) Model clearly shows a northern limit forAe.albopictus, particularly in northern Honshu in 2035 and southern and middle Hokkaido Island in 2100 in Japan. The spread of the mosquito vector through global used-tire trading in recent decades to Africa, the Mideast, Europe, and North and South America caused an outbreak of Chikungunya fever in north Italy in 2007. Global warming, extreme climate change, changing physical distribution, and an increase in oversea travel are also expected to influence the epidemiology of vector-borne infectious diseases.

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 107/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.

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 a10% 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 ratio2,307), 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.

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.